This study investigated the extracts of Euphorbia hirta and Polygonum plebeium against human bacterial pathogen Pseudomonas. aeruginosa PAO1, B24 (isolated from wound infection of the arm, Cleopatra Hospital) with accession no.0748281. Among all tested solvents, ethyl acetate extract of Euphorbia hirta and P.plebeium was the most effective. Also the ethyl acetate extract of both medicinal plants has synergitic effect which increased the antimicrobial activity against P. aeruginosa PAO1, B24. MIC of plant extract P.plebeium and E.hirta against the growth of P. aeruginosa PAO1, B24 was found to be 12.5mg/m. Parameters controlling the antimicrobial activity of ethyl acetate extract of E.hirta and P.plebeium had been studied. It was more effective when incubated at 45°C for microbial strains P. aeruginosa PAO1, B24; pH5 was more effective against P. aeruginosa PAO1, B24; Dark condition for 24hr was effective against P. aeruginosa PAO1, B24; Static condition had slight effectiveness; Xylose exhibited the highest antimicrobial activity against P. aeruginosa PAO1, B24; it was not affected by the different types of amino acids and no metallic ions was effective against P. aeruginosa PAO1, B24. Also no vitamins were effective on the microbial strain under study.