Pseudomonas aeruginosa was used to synthesize anisotropic gold nanoparticles from the unusually reducible aryldiazonium gold (III) salt of the chemical formula [HOOC-4-C6H4N≡N]AuCl4 (abbreviated as DS-AuCl4). We investigated the effect of bacterial cell density, temperature, and pH on the AuNP synthesis. The bacterial cell density of 6.0 × 108 CFU/mL successfully reduced 0.5 mM DS-AuCl4 salt to AuNPs after incubation at 37 °C (24 h), 42 °C (24 h), and 25 °C (48 h). Transmission electron microscopy (TEM) images revealed the formation of spherical, triangle, star, hexagon, and truncated triangular morphologies for the AuNPs synthesized using P. aeruginosa bacteria. The average size of AuNPs synthesized at 25 °C (48 h), 37 °C (24 h), and 42 °C (24 h) was 39.0 ± 9.1 nm, 26.0 ± 8.1 nm, and 36.7 ± 7.7 nm, respectively. The average size of AuNPs synthesized at pH 3.7, 7.0, and 12.7 was 36.7 ± 7.7 nm, 14.7 ± 3.8 nm, and 7.3 ± 2.5 nm, respectively, with the average size decreasing at a pH of 12.7. The reduction of the DS-AuCl4 salt was confirmed using X-ray photoelectron spectroscopy (XPS). The significant peaks for C1s, Au4f doublet, N1s, and O1s are centered at 285, 84–88, 400, and 532 eV. The ability of inactivated bacteria (autoclave-dead and mechanically lysed bacteria), peptidoglycan, and lipopolysaccharides to reduce the DS-AuCl4 salt to AuNPs was also investigated. Anisotropic AuNPs were synthesized using inactivated bacteria and peptidoglycan but not using lipopolysaccharides. The AuNPs demonstrated biocompatibility with human RBCs and were safe, with no antibacterial activities against Escherichia coli and Staphylococcus aureus. This is the first report demonstrating the synthesis of AuNPs using aryldiazonium gold(III) salts with P. aeruginosa. These AuNPs are promising candidates for exploring potential applications in nanomedicine and drug delivery.