A series of novel substituted 2-((3-chloro-2-methylphenyl) ((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino) benzoic acid (5a-l) derivatives were synthesized using a multi-step synthetic route involving the click chemistry approach. The structures of the synthesized compounds were confirmed using various spectroscopic techniques, including NMR, IR, Mass and elemental analysis. The antibacterial and antifungal activities of the synthesized compounds were evaluated against a panel of bacterial strains (including B. Subtilis, B. Sphaericus, and S. Aureus, P. Aeruginosa, K. Aerogenes and C. violaceum) and fungal species (such as C. albicans, A. Fumigatus, T. Rubrum and T. mentagrophytes). The results revealed significant inhibitory effects, with some derivatives showing superior activity compared to standard drugs. Additionally, the cytotoxicity of these compounds was assessed against human cancer cell lines (MCF-7, PC-3, and HeLa) using MTT assays. Several derivatives exhibited potent cytotoxic effects, indicating their potential as anticancer agents. Molecular docking studies of newly synthesized derivatives 5a-l, along with Doxorubicin and Tamoxifen, against enoyl reductase (PDB ID: 1QSG) indicate potential inhibitors for lipid biosynthesis in cancer therapy.