In the present study, the crude secondary metabolites, previously extracted from the fermentation medium of the locally isolated Pseudomonas aeruginosa OQ158909 strain were subjected to silica gel column chromatography using optimized solvent systems. The most biologically active fraction was further purified via high-performance liquid chromatography (HPLC) and identified as phenazine-1-carboxylic acid (PCA) through a comprehensive analytical approach involved ultraviolet (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, mass spectrometry (MS), and proton nuclear magnetic resonance (¹H-NMR) spectroscopy. The biological activities of the purified PCA were evaluated, revealing a potent antimicrobial effect against a diverse array of human pathogens. The inhibition zones recorded against Gram-positive bacteria ranged from 16 to 28 mm, while those against Gram-negative bacteria ranged from 13 to 36 mm. PCA also exhibited antifungal activity, with inhibition zones of 16 mm and 17 mm against Aspergillus niger and Candida albicans, respectively. Furthermore, PCA demonstrated significant antioxidant activity, scavenging 75.2% of DPPH free radicals at a concentration of 100 μg/mL, with an IC₅₀ value of 40.4 μg/mL. This efficacy surpassed that of butylated hydroxyanisole (BHA) and vitamin C, which served as standard positive controls in the antioxidant assays.In addition, PCA exhibited pronounced anti-inflammatory effects. Treatment with 100 μg/mL of PCA resulted in an 82.54% reduction in lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nearly complete suppression of inducible nitric oxide synthase (iNOS) expression. PCA also displayed significant cytotoxic effects against various human cancer cell lines. The highest efficacy was detected against the HePG2 cell line, with an IC₅₀ of 45.5 μg/mL and a selectivity index exceeding 2. Moreover, 100 μg/mL of PCA inhibited 74% of PACA cells and 50% of PC3 and MCF7 cells. Lastly, PCA showed remarkable wound healing potential, achieving nearly complete wound closure at a concentration of 100 μg/mL within 24 hours.