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The biofilm of drug-resistant pathogenic bacteria is a serious issue worldwide. The natural growth of blue-green algae Microcystis sp. has the potential to antagonize the bacterial pathogens in a novel approach. In the present study,Microcystis sp. was isolated and its mass production was optimized by Response Surface Methodology (RSM) and also, the invitro antioxidant and antibiofilm potentials against the drug-resistant pathogens were investigated. The experimental design was carried out on mass production of Microcystis sp. with pH, and mineral salts as independent variables.The maximum biomass yield (1.5 g/L) was achieved with NaNO3 (20mM) andK2HPO4 (10mM) at pH 7.5. The Microcystis sp. exhibited remarkable antioxidant (65%) and antibiofilm activities against Staphylococcus aureus and Pseudomonas aeruginosa. The Gas chromatography-mass spectrometry (GC-MS)of Microcystis sp. extract analysis revealed the presence ofsixteen bioactive potential compounds which proved the synergistic activity against the bacterial pathogens. Therefore, the potential bioactive compoundsinMicrocystis sp. could be an effective biocontrol agentagainst the challenging drug-resistant bacterial pathogens.