A total of 65 blue-green pigment-producing bacterial strains were isolated from different environmental sources. The strains were identified as Pseudomonas aeruginosa Z57 and Pseudomonas aeruginosa Z62 using the Vitek2 identification system. Both strains were confirmed to be Gram-negative and exhibited positive reactions for several enzymes, including glutamyl arylamidase, d-glucose, gamma-glutamyl-transferase, d-mannose, beta-alanine arylamidase, and proline arylamidase, while testing negative for others. Optimal growth conditions were established at 35°C and pH 7, utilizing glucose and casein as carbon and nitrogen sources, respectively. The most potent isolate, Pseudomonas aeruginosa WM1, was confirmed by 16S rRNA sequencing and registered in Gene Bank with Accession No. (PV055704). Pyocyanin was extracted, and then purified using column chromatography. It was further characterized by UV–Vis Spectrophotometer, Fourier transformer Infrared spectrometry (FTIR), and nuclear magnetic resonance (NMR). Antimicrobial assays showed that higher concentrations of purified pyocyanin resulted in larger inhibition zones against pathogenic microbes. Interestingly, bacterial strains demonstrated greater resistance to pyocyanin than fungal strains; at a concentration of 2.5 µg/mL, Staphylococcus aureus and Aspergillus niger were identified as the most resistant organisms.