Nosocomial infections caused by multiple antibiotic resistant bacteria that are particularly difficult to cure, cause a considerable health risk and place an enormous burden on the economy. A primary reason of such nosocomial infections is the biofilm-forming Pseudomonas aeruginosa that primarily infects immunocompromised individuals and those with severe burn wounds. Inspite of P. aeruginosa being among the major cause of nosocomial infections, still little is known about the in vivo biofilm phenotype and the bacterial factors that prevent wound healing and promote persistence of P. aeruginosa at the site of infection. In the present study, P. aeruginosa isolates were collected from surgical wards of a tertiary care hospital of Pakistan Institute of Medical Sciences, Islamabad, Pakistan, from November 2007 to February 2009. The isolates were identified as P. aeruginosa on the basis of colony characteristics, Gram‘s staining, oxidase, catalase, gelatin hydrolysis and sugar fermentation test and further confirmed by culturing on selective medium, i.e., Pseudomonas cetrimide agar. Eight of the clinical isolates were further reconfirmed by polymerase chain reaction (PCR), by amplifying 16S rRNA gene. Antimicrobial susceptibility of all 102 P. aeruginosa isolates was performed by the Kirby-Bauer method and results were deduced according to National Committee for Clinical Laboratory Standards (NCCLS) criteria. The antibiotics tested included: ciprofloxacin, gentamicin, amikacin, tobramycin, piperacillin, ceftazidime, cefoperazone, cefotaxime, imipenem, and meropenem. Exopolysaccharide production by the clinical isolates was checked by Congo red assay and pellicle production was monitored by visual examination of the air-liquid interface. Swimming ability of the selected isolates was checked by using 0.3% agar. Group behaviour of eight clinical isolates was observed during biofilm formation, twitching motility, swarming motility and rhamnolipid production. pelA mutation in 2 clinical isolates of P. aeruginosa (i.e., strain 99 and strain 160) was generated by a single-crossover (SCO) insertion using the pMQ89 suicide vector. Minimal bactericidal concentration (MBC) for biofilm and planktonic cells of Pseudomonas aeruginosa was calculated by using a 96 well microtitre plate assay. The distribution of total 102 P. aeruginosa isolates 23 (22.55%) and 79 (77.45%) of P. aeruginosa isolates were isolated from females and males, respectively. The resistance to antibiotics was gentamicin 65.7%, amikacin 55.9%, tobramycin 63.7%, ciprofloxacin 54.9%, imipenem 25.5%, meropenem vi29.4%, ceftazidime 77.5%, cefoperazone 76.5%, cefotaxime 98%, piperacillin 64.7%. Therefore, carbapenems (i.e., imipenem and meropenem) were found as the most affective drug against P. aeruginosa in the present study. In the present study, an increasing trend of antimicrobial resistance was observed. The major reason for such a high resistance was due to misuse of antibiotics in key areas of the hospital. The main factor responsible for the misuse of antibiotics was misdiagnosis of multidrug- resistant organisms. Most of the isolates showing multidrug resistance were polysaccharide producers, i.e., they were showing red color on Congo red agar plates. Biofilm forming strains were mostly not found to be showing high antibiotic resistance. All the isolates showed swimming motility. One of the isolate, i.e., 99 was a hyperbiofilm former while all of the other isolates tested showed less or almost equal biofilm formation to the lab strain PA14. All isolates were positive for swarming motility while for twitching motility only two gave negative result (i.e., strain 2 and 113). All the tested isolates were rhamnolipids producers except 2 of them, i.e., strain 99 and strain 47. As observed for lab strain PA14, all the eight clinical isolates showed higher values of MBC for biofilm as compared to their planktonic values against the two classes of antibiotic tested, i.e., aminoglycosides and fluroquinolones. Deletion of pel gene from PA14 resulted in a reduction of biofilm formation. PelA mutations in 2 of the clinical isolates also showed decreased biofilm formation as compared to their parent strain but this reduction was not significant. Little information is present about the function of different components of the matrix in biofilm-associated antibiotic resistance. It was found that the existence of exopolysaccharide decreased the extent of biofilm-associated antibiotic resistance against one class of antibiotics. The MBC of biofilm-grown ΔpelA mutant of PA14, which does not manufacture Pel polysaccharide, was 4-fold elevated in case of tobramycin and gentamicin, and was unaltered for ΔbifA mutant, which overproduced Pel polysaccharide, as compared to the wildtype. Biofilms of pelA mutants in 2 clinical isolates of Pseudomonas aeruginosa exhibited 8-fold higher MBC for tobramycin as compared to wildtype. No difference was observed in the biofilm resistance of any of these strains when tested against fluoroquinolones. The present work will serve as a base for further studies enlightening the mechanisms of biofilm- associated antibiotic resistance against aminoglycosides by P. aeruginosa.