Various analogues of benzimidazole are found to be biologically and therapeutically potent against several ailments. Benzimidazole when attached with heterocyclic rings has shown wide range of potential activities. So, from the above provided facts, we altered benzimidazole derivatives so that more potent antagonists could be developed. In the search for a new category of antimicrobial and anticancer agents, novel azomethine of 2-mercaptobenzimidazole derived from 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzohydrazide were synthesized.The synthesized analogues were characterized by FT-IR, 1H/13C-NMR and MS studies as well C, H, N analysis. All synthesized compounds were evaluated for in vitro antibacterial activities against Gram-positive (B. subtilis), Gram-negative (E. coli, P. aeruginosa, K. pneumoniae and S. typhi) strains and in vitro antifungal activity against Candida albicans and Aspergillus niger strains by serial dilution method, the minimum inhibitory concentration (MIC) described in µM/ml. The in vitro anticancer activity of synthesized compounds was determined against humancolorectal carcinoma cell line (HCT-116) using 5-fluorouracil as standard drug.In general, all the synthesized derivatives exhibited significant antimicrobial and anticancer activities. Compounds 8, 10, 15, 16, 17, 20 and 22 showed significant antimicrobial activity towards tested bacterial and fungal strains and compound 26 exhibited significant anticancer activity.