Synthesis and evaluation of cytotoxicity and anti-microbial activity of a series of 1,4-bis(4-substituted-5-mercapto-1,2,4-triazol-3-yl)butane derivatives comprising thioether functionality and other pharmacophore modifications are described. All the newly synthesized compounds were characterized by IR, NMR, elemental analyses, and mass spectral studies. The compounds 4a–f, 5a–f, and 6a–f were evaluated for in vitro cytotoxicity potential using the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against a panel of three human cancer cell lines: Lung carcinoma A-549, Colon carcinoma HT-29, and Breast Cancer MDA MB-231. All the compounds were subjected to in vitro antibacterial activity against Bacillus subtilus (ATCC 6633), Staphylococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), and Pseudomonas aeruginosa (ATCC-27853) and their minimal inhibitory concentrations were determined.