The present studies were aimed to isolate and characterize the secondary metabolites responsible for antibacterial activity from medicinal plants present in Pakistan. Two plant species Dodonaea viscosa (L.) Jaeq. and Quercus baloot Griff. were selected on the basis of literature review and their traditional uses in ailments related to microorganisms. The n-hexane, dichloromethane, ethyl acetate, n-butanol and aqueous fractions of Dodonaea viscosa were analyzed for antimicrobial potential against four Gram positive bacteria: Bacillus subtilis (MRL M 1), Bacillus cereus (MRL M 52), Micrococcus luteus (ATCC 10240), Staphylococcus aureus (ATCC 6538); three Gram negative bacteria: Escherichia coli (ATCC 25922), Salmonella typhi (Cl. I. 140), Pseudomonas aeruginosa (ATCC 9721) and the yeast Candida albicans (Cl. I. 4043). It showed inhibition against S. aureus, M. luteus, B. subtilis, E. coli, P. aeruginosa and C. albicans. The TLC solvent systems for each of the fraction were developed and the resulting chromatograms of the fractions were sterilized using ethylene oxide or dioxane, which were then subjected to contact bioautography. Multiple inhibition zones were observed at different R f values against B. subtilis, M. luteus, E. coli, S. typhi, P. aeruginosa, and C. albicans indicating the presence of antimicrobial components. Isolation of the active principles responsible for the antimicrobial activities was attempted through preparative TLC, but it was unable to yield the compounds. The results from the preliminary screening and contact bioautography indicated n-hexane, ethyl acetate and n-butanol fractions having good potential in terms of antimicrobial activities, therefore, they were chosen for further investigations. HPLC revealed the presence of large number of metabolites; therefore, further isolation was done using preparative HPLC that resulted in 52 sub-fractions each for n-hexane and n-butanol fractions while 48 sub-fractions were obtained from ethyl acetate fraction. XTT-Bioassay was used in hyphenation with preparative HPLC to mark the antibacterial potential of the emerging sub-fractions. S. aureus (NCIMB 6571) and E. coli (NCIMB 8797) were used in all XTT based bioassays. On the basis of bioassay results, six sub-fractions from n-hexane fraction were selected and analyzed upon HPLC in analytical mode, which indicated multiple numbers of compounds in them, thereby, necessitating further isolation. Further fractionation gave 218 sub-sub fractions that were tested against the same two bacteria. The sub-sub fractions indicating antibacterial activity were analyzed upon HPLC and isolation of the compound was possible from the sub-sub fraction no. 12 of sub-fraction 42 of n-hexane fraction of D. viscosa’s crude ethanolic extract. The compound’s MIC’s against S. aureus (NCIMB 6571) and E. coli (NCIMB 8797) were 64 μg/ml and 128 μg/mlxviii respectively. The MBC’s against these organisms were 128 μg/ml and 256 μg/ml, respectively, which indicated a moderate activity against the Gram-positive bacterium. The structure analyses revealed the compound to be 15, 16-epoxy-cis-cleroda-3, 13(16),14- trien-18-oic acid-18,6-olide, a clerodanefuranolactone, previously known for its structure but this is the first report of its antibacterial potential and its presence in D. viscosa. Quercus baloot fractions were processed in the same manner and were subjected to antimicrobial analysis using similar panel of microorganisms. Preliminary screening using disk diffusion and agar well diffusion methods showed inhibition zones against S. aureus, M. luteus, B. subtilis, E. coli, P. aeruginosa and C. albicans. TLC chromatograms and subsequent contact bioautography showed inhibition zones at different R f values against B. subtilis, M. luteus, E. coli, and S. aureus indicating the presence of antimicrobial components. On the basis of these findings QDM fraction underwent HPLC evaluations that indicated a good number of metabolites; therefore, preparative HPLC was carried out that yielded 52 sub-fractions that were subjected to XTT bioassay to mark the antibacterial potential from which five potential sub-fractions were again analyzed upon HPLC. Each sub-fraction had several compounds, thereby; preparative HPLC was applied that resulted in 175 sub-sub fractions, which were subjected to XTT bioassay using same two bacteria. The sub-sub fractions indicating antibacterial activity were analyzed upon analytical HPLC and isolation of a semi-purified compound was made from the sub-sub fraction no. 15 and 16 of sub-fraction 39 of dichloromethane fraction obtained from Q. baloot’s crude methanolic extract. Its MIC’s against S. aureus (NCIMB 6571) and E. coli (NCIMB 8797) were 16 μg/ml and 128 μg/ml respectively while MBC’s were 64 μg/ml and 256 μg/ml, respectively. This compound requires further purification and characterization. This is the first report of such an activity in Q. baloot.