Passiflora is a genus belonging to the family Passifloraceae having varied species with highly therapeutic values. Tribes with traditional medicinal knowledge suggested Passiflora foetida L. as a source of high value pharmaceutical plant. Present study deals with the Isolation, Purification, characterization and bioassays of antimicrobial secondary metabolites. In spite of traditional soxhlet extraction, Cold percolation proved suitable extraction scheme. Bioassay guided TLC characterization, and purification led the effective collection of bioactive natural products. Disc diffusion method shows a potent inhibitory activity of compounds against multi drug resistant pathogenic microorganisms like S. aureus (OARS). For structural characterization bioactive products were analyzed using UV. The results confirmed the presence of polyacetylenes as active constituents in the plant.