Based on the biologically active heterocycle compounds, a series of new benzimidazole-hydrazone derivatives (3a-3j) were synthesized, starting from 4-(6-chloro-1H-benzimidazol-2-yl) benzohydrazide. The synthesized compounds were characterized by 1H NMR, 13C NMR, and HRMS spectroscopic methods. The synthesized compounds were preliminarily evaluated for their in vitro antimicrobial, anticancer and antioxidant activity. The antimicrobial activity was checked against S. aureus ATCC 29213, E. coli ATCC 25922, and C. albicans ATCC 10231 by micro-dilution method. The findings exhibited that the compounds possessed moderate antimicrobial potential. The compounds were also checked for their in vitro anticancer activities against HT-29 (colorectal cancer cell line) using the MTT assay. It was observed that all the compounds 3a-3j showed weak antiproliferative activity against HT-29 cells. The compounds were also analyzed for their antioxidant capacity by Tas & Tos activity. The compound 3d showed a high anti-oxidative effect with 30.81 µmol H2O2 Equiv./L value. The lowest energy state of compound 3d was realized in DMSO medium by using the B3LYP method at 6–311G (d,p) level, the optimized geometry of it is about 0.50 and 17 kcal/ mol more stable than in other solvents and the gas phase, respectively. The lower ΔE=3.563 eV indicates the higher reactivity of compound 3d, this is compatible with biological experimental data and shows high antioxidant property of the compound. In silico ADMET studies of compound 3d were performed. Indexing of the X-ray powder diffraction pattern was performed for compound 3a.