Dihydrofolate reductase (DHFR) is an important target for antimetabolite class of antimicrobials because it participates in purine synthesis. 2-mercaptobenzimidazole (2MBI) has similar structural features as purine nucleotides. Given that benzimidazole and similar heteroaromatics have been broadly examined for their anticancer potential, so, we hereby report the design, synthesis and biological studies (i.e. antimicrobial and anticancer studies) of 2MBI derivatives.The antimicrobial activity of synthesized 2MBI derivatives were evaluated against Gram positive and Gram negative bacterial species as well as fungal species by tube dilution technique whereas their anticancer activity was assessed against human colorectal carcinoma cell line (HCT116) by Sulforhodamine B (SRB) assay. They were also structurally characterized by IR, NMR, MS and elemental analyses.The antimicrobial activity findings revealed that compound (MIC  = 1.27, 2.54, 1.27 µM), (MIC = 1.43 µM), (MIC = 2.60 µM), and (MIC = 2.65 µM) exhibited significant antimicrobial effects against tested strains, i.e. Gram-positive, Gram-negative (bacterial) and fungal strains. The anticancer screening results demonstrated that compounds , (IC = 5.85, 4.53 µM) were the most potent compounds against cancer cell line (HCT116) even more than 5-FU, the standard drug (IC = 9.99 µM).