HDAC6 is a crucial epigenetic modifier that plays a vital role in tumor progression and carcinogenesis due to its multiple biological functions. It is a unique member of class-II HDAC enzymes. It possesses two catalytic domains, which function independently of the overall enzyme activity. Up to date, there are only a few selective HDAC6 inhibitors with anti-cancer activity. In this study, 175,204 ligands obtained from the ZINC15 and OTAVAchemical databases were used for virtual drug screening against HDAC6. Molecular docking studies were performed for 100 selected compounds. Furthermore, the top 10 compounds obtained from docking were tested for their efficacy to inhibit the function of HDAC6. Five compounds (N-(9-oxo-9H-fluoren-3-yl)benzamide, 2-hydroxy-5-[(5-oxo-6-phenyl-4,5-dihydro-1,2,4-triazin-3-yl)amino]benzoic acid, 5-(4-bromonaphthalene-1-sulfonamido)-2-hydroxybenzoic acid, 2-(naphthalen-2-yl)-N-(1H-1,2,3,4-tetrazol-5-yl)cyclopropane-1-carboxamide, and 4-oxa-5,6 diazapentacyclo[10.7.1.0³,⁷.0⁸,²⁰.0¹⁴,¹⁹]icosa-1,3(7),5,8(20),9,11,14,16,18-nonaen-13-one) inhibited enzymatic activity by more than 50 % compared to DMSO as the control. Two candidates, (N-(9-oxo-9H-fluoren-3-yl)benzamide and 5-(4-bromonaphthalene-1-sulfonamido)-2-hydroxybenzoic acid), were identified with considerable cytotoxicity towards drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Microscale thermophoresis revealed the binding of N-(9-oxo-9H-fluoren-3-yl)benzamide and 5-(4-bromonaphthalene-1-sulfonamido)-2-hydroxybenzoic acid to purified HDAC6 protein. Both compounds induced apoptosis in a dose-dependent manner as analyzed by flow cytometry. In conclusion, we demonstrate for the first time that these two compounds bind to HDAC6, inhibit its function, and exert cytotoxic activity by apoptosis induction.