The aim of this work was to investigate the antimicrobial effects of biosurfactant (rhamnolipid) produced from Pseudomonas sp. Eight clinical test microorganisms were chosen, which were different groups, for the antimicrobial assays. Antimicrobial activity was evaluated according to the minimal inhibitor concentration (MIC) and disc-diffusion method. The highest activity for the rhamnolipid discs was obtained for beta-hemolytic Streptococcus sp., whereas the lowest activity was found for Pseudomonas aeruginosa. The biosurfactant (rhamnolipid) showed very strong antimicrobial activity against the microorganisms tested.