Pyocyanin is the characteristic blue-green phenazine pigment produced by Pseudomonas aeruginosa stra- ins. The aim of our study is to determine the highest amount of pyocyanin production in P. aeruginosa strains and to investigate the antimicrobial effect of pyocyanin pigment on Escherichia coli, Bacillus sp., Pseu- domonas aeruginosa, Candida sp. and Aspergillus niger. For this purpose, pyocyanin was extracted from P. ae- ruginosa culture supernatants and purified, and then it was measured by spectrophotometrically. P. aerugino- sa A10 strain isolated from urine specimen was determined as the highest amount of pyocyanin producer and P. aeruginosa A1 strain isolated from abdomen was the lowest. The ‘Agar Well Diffusion’ method was carried out to determine the antimicrobial effect of pyocyanin. Accordingly, pyocyanin was effective as an antimicrobial agent on Bacillus sp., Escherichia coli and Candida sp., but there was no antimicrobial effect on P. aeruginosa and A. niger. After that ‘Tube Dilution Method’ was used on strains, which were determined as sensitive to pyocyanin with agar well diffusion method. An increase in the intensity of Bacillus sp. and Candida sp. strains’ growth with decreasing the concentration of pyocyanin was observed.