Pyocyanin is a bioactive pigment produced by Pseudomonas aeruginosa. It is an important virulence factor that plays a critical role in P. aeruginosa infections as a redox-active secondary metabolite and a quorum sensing (QS) signaling molecule. Pyocyanin production from chorismic acid requires the involvement of two homologous operons, phz1 and phz2, which are activated by QS regulatory proteins. Pyocyanin inhibits the proliferation of bacterial, fungal, and mammalian cells by inducing oxidative stress due to which it acts as a potent antibacterial, antifungal, and anticancer agent. Its potential role as a neuroprotectant needs further exploration. However, pyocyanin exacerbates the damaging effects of nosocomial infections caused by P. aeruginosa in immunocompromised individuals. Further, cystic fibrosis (CF) patients are highly susceptible to persistent P. aeruginosa infections in the respiratory system. The bacterial cells form colonies and three interconnected QS networks-pqs, las, and rhl-get activated, thus stimulating the cells to produce pyocyanin which exacerbates pulmonary complications. As an opportunistic pathogen, P. aeruginosa produces pyocyanin to impede the recovery of injuries like burn wounds through its anti-proliferative activity. Moreover, pyocyanin plays a vital role in compounding P. aeruginosa infections by promoting biofilm formation. This review begins with a brief description of the characteristics of pyocyanin, its activity, and the different aspects of its production including its biosynthesis, the role of QS, and the effect of environmental factors. It then goes on to explore the potential applications of pyocyanin as a biotherapeutic molecule while also highlighting the biomedical challenges and limitations that it presents.© 2024. The Author(s).