New benzimidazole analogs were prepared and tested for antimicrobial efficacy toward Escherichia coli ATCC 12435, Bacillus cereus UW 85, Staphylococcus aureus ATCC 29213, Candida albicans and Aspergillus fumigatus 293. Results indicated that compound 10 has potent and broad spectrum antimicrobial activity. In addition, 4b and 5c showed eminent antimicrobial efficacy toward B. cereus, S. aureus, C. albicans and A. fumigatus. Furthermore, 12 and 14 demonstrated interesting antifungal activity toward C. albicans. Antiquorum-sensing efficacy of the new compounds toward Chromobacterium violacium ATCC 12472 was also examined. In vitro antitumor screening of the new benzimidazoles toward HepG2, HCT-116 and MCF-7 cancer cell lines demonstrated that 4b and 5b,c are the most potent analogs toward all tested cell lines. The three potent in vitro antitumor analogs were further assessed for in vivo antitumor activity toward EAC in mice, and in vitro cytotoxicity toward W138 normal cell line. Results revealed that 4b has the highest in vivo activity, and that the three tested analogs are less cytotoxic than 5-FU toward W138 normal cell line. The most active antimicrobial and antitumor analogs were examined for DNA-binding affinity, whereas 4b and 5c displayed the highest affinity. The in silico studies illustrated that all of the new benzimidazoles meet the optimal requirements for good oral absorption and bioavailability. Moreover, in silico toxicity assessment has been reported.