New benzimidazole derivatives were synthesized and assessed for antimicrobial efficacy toward Escherichia coli, Bacillus cereus, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus 293. Results indicated that compounds 3c and 3n have promising activity toward S. aureus, whereas 3i exhibited remarkable efficacy toward B. cereus. Moreover, compound 3c was proved to be the most active antifungal analog toward C. albicans. On the other hand, 3n displayed the highest activity against A. fumigatus 293. Antiquorum-sensing activity of the same compounds was also tested against Chromobacterium violacium ATCC 12472, whereas compounds 3c-f, 3i-k and 3m-o showed acceptable activity. In vitro antitumor testing of these compounds toward liver cancer (HepG2), colon cancer (HCT-116) and breast cancer (MCF-7) cell lines revealed that compound 3p has the highest potency against the three tested cell lines. Moreover, 3f, 3m and 3n displayed promising activity toward all tested cell lines. Compounds 3f, 3m, 3n and 3p were esteemed for in vivo antitumor activity against EAC cells. The active antimicrobial and antitumor analogs, 3a, 3c, 3f, 3i-k, 3m, 3n and 3p were assessed for DNA-binding affinity, and results indicated that 3c, 3f, 3i, 3k and 3n have strong DNA-binding affinity. The computational studies affirmed that almost all of the inspected compounds meet the optimal requirements for good absorption and oral bioavailability.