Novel Mannich bases derived from 2-substituted benzimidazole and (thio) hydantoin moieties, were synthesized as histone deacetylase 6 (HDAC6) inhibitors with potential cytotoxic activity. All derivatives were tested In vitro against HDAC6 enzyme. IR, 1H NMR, 13C NMR and mass spectroscopy confirmed the structure of synthesized compounds. All tested compounds significantly inhibited HDAC6 at nanomolar level. Compound 2c was the most potent presenting significant HDAC6 inhibitory activity (IC50 = 97.356± 5.7 nM), nearly equipotent to SAHA reference drug (IC50 = 91.732 ± 5.4 nM). In vitro cytotoxicity study was also carried out. Compound 2c exhibited potent cytotoxic activity against the tested cells (CCRF-CEM and MOLT-4) showing one digit micromolar IC50s. Compound 2c (IC50 = 3.66±0.22 uM) was 2-fold more active than SAHA reference drug (IC50 = 6.8±0.41 uM) against MOLT-4 cell line. Furthermore, a docking study demonstrated the ability of target Mannich bases to achieve an excellent fitting inside the binding site of HDAC6 enzyme.