Treatment of infectious diseases has become difficult because of the emergence of multi-drug resistant bacteria. Main reasons of antibiotic resistance include the irrational use of broad spectrum antibiotics, prescription of antibiotics without doing culture tests, self medication because of over the counter availability of the antibiotics and a long hospital stay. This study was designed to determine the frequency of multi drug resistant (MDR) isolates among clinical isolates from in and out-patients at the Ayub Teaching Hospital (ATH), Abbottabad, characterize the infection related isolates phenotypically by two methods, disc diffusion and Minimum Inhibitory Concentration (MIC) and evaluate whether the different methods are efficient tools in routine epidemiological investigations. A total of 405 samples were sent to the microbiology section of the Ayub Teaching Hospital for culture and sensitivity. Among these 68.64% samples showed bacterial growth which included Escherichia coli, Staphylococcus aureus, Proteus species, Klebsiella species, Pseudomonas aeruginosa, Enterococcus species and streptococcus species. Gram negative bacteria were the most prevalent group with percentage of 56.12%, while Gram positive was 43.88%. The most prevalent bacteria were E. coli (41.7%) followed by S. aureus (35.25%). P. aeruginosa (8.27%), Streptococcus spp. (7.19%), Proteus spp. (5.03%), Enterococcus spp. (1.43%), and Klebsiella spp. (1.07%). The samples including urine, pus, blood etc received for culture and sensitivity were further processed according to standard microbiological techniques. The properly identified strains were subjected to antimicrobial testing by the modified KirbyBauer Disc diffusion method and MIC method following the Clinical Laboratory and Standards (CLSI) guidelines. The commonly used antibiotics including; amoxicillin, amoxicillin+clavulanic acid, cephradine, ceftazidime, cefoperazone+sulbactam, ceftizoxime, cefuroxime, ceftriaxone, imipenem, meropenem, erythromycin, cefaclor, ciprofloxacin, gentamycin, trimethoprim+sulfamethoxazole were employed for testing. 67.92% of S. aureus were isolated from pus, 10.96% from urine, and 38.46% from miscellaneous samples. 24.49% isolates were found to be Methicillin resistant S. aureus (MRSA) 75.51% were Methicillin sensitive S. aureus (MSSA). Among MSSA, 37.84% were found to be multi drug resistant (MDR). All the 24 MRSA were also found to be MDR. In total, 53.06% isolates were found to be MDR. The most potent antibiotic was imipenem with 94.90% activity followed by cefoxitin and trimethoprim+sulfamethoxazole. All MRSA strains were 100% sensitive to imipenem. Resistance shown to cephalosporins ranged from 50 to 83.33%. Resistance was high in MRSA as compared to MSSA. MSSA like MRSA were all 100% resistant to amoxicillin, 74.32 % to ceftazidime and 59.46% to cefaclor. These MSSA were sensitive to imipenem (93.24%), Trimethoprim+sulfamethoxazole (68.92%), and amoxicillin+clavulanic acid (51.35%). MRSA were resistant to multiple antibiotics. In case of E. coli high resistance was observed for cephradine (73.28%) followed by cefuroxime, ceftazidime, meropenem and erythromycin. Imipenem was the most efficient antibiotic. MIC studies confirmed that cephradine is the most resistant of all antibiotics tested and imipenem is the most effective of all antibiotics tested. The most effective antibiotic against P. aeruginosa was imipenem, and high resistance was observed against erythromycin and cefuroxime (100%). MIC studies showed that amoxycillin+clavulanic acid was the most effective antibiotic with 65.22% susceptibility and erythromycin (100%) is the most resistant of all antibiotics tested. Streptococcus spp. showed maximum resistance against meropenem (100%) followed by ceftriaxone and cephradine. Imipenem showed excellent activity (100%). MIC studies found that cefepime, meropenem and ceftriaxone are the most resistant of all tested antibiotics. Imipenem and trimethoprim+sulfamethoxazole were most effective having 90% susceptibility. Proteus spp. showed maximum resistance against cephradine (85.71%) followed by cefuroxime and amoxicillin+clavulanic acid. Imipenem and ceftriaxone showed excellent (100%) activity against the Proteus spp. MIC studies showed that ceftizoxime was most effective while gentamycin, cephradine and meropenem are the most resistant of all tested antibiotics. Enterococcus spp. showed maximum resistance against cephradine, cefaclor and meropenem. MIC studies confirmed that cephradine was most resistant of all tested antibiotics. Ciprofloxacin, ceftriaxone, gentamycin, cefoperazone+sulbactam, amoxicillin and imipenem showed 25% resistance. Klebsiella spp. showed 100% resistance against cephradine, cefuroxime and amoxicillin and 100% sensitivity to cefaclor, meropenem, ceftazidime, amoxicillin+clavulanic acid, trimethoprim+Sulfamethoxazole, ciprofloxacin, ceftriaxone, gentamycin, ceftizoxime and imipenem. MIC studies indicated that amoxycillin was most resistant of all antibiotics and all the Klebsiella spp. were sensitive to ceftizoxime, trimethoprim+sulfamethoxazole, ciprofloxacin, cefaclor, gentamycin and imipenem. In this study 65.83% isolates were identified as MDR Gram positive and Gram negative microorganisms. 53.55% of MDR isolates were obtained from urine, 38.25% from pus, 1.64% from High vaginal swab (HVS) and 6.56% from miscellaneous samples. E. coli was the most frequent (40.98%) multi-drug resistant isolate in the current study followed by S. aureus (28.42%), P. aeruginosa (11.48%), Strep spp. (10.38%), Proteus spp. (6.01%), Klebsiella spp. (1.64%), and Enterococcus spp. (1.09%). The study revealed that antibiotic resistance has become a significant problem and will continue as bacteria continue to evolve under the selective pressure of antibiotics. Unfortunately, the misuse of these life saving medications, coupled with bacteria’s amazing ability to adapt, has led to an increase in the number of drug-resistant organisms. In response, scientists are scrambling to develop new drugs. The most important steps in preventing nosocomial infections are to first recognize their occurrence and then establish policies to prevent their development. Slowing the emergence and spread of bacteria involves the cooperation of health care personnel, educators, and the general public. Physicians should prescribe antimicrobial medications only when appropriate. The public must be educated about the appropriateness and limitations of antimicrobial therapy. Patients need to carefully follow prescribed instructions when taking antimicrobials.