A series of novel β-carboline 1,3,4-oxadiazole based hybrids were designed, synthesized, and tested for cytotoxicity and HDAC inhibition. Among the target compounds, compound ZDLT-1 displayed high inhibitory activity for class I HDACs 1, 2, and 3, and potent anti-proliferative activity against HCT116 cells with an IC<sub>50</sub> value of 0.173 ± 0.018 μM, it also exhibited better inhibitory activity with an IC<sub>50</sub> value of 6 nM for HDAC6 than SAHA (IC<sub>50</sub> = 15 nM). Furthermore, the pharmacological experiment of Hoechst staining, colony formation, cell apoptosis assay, and wound healing scratch assay indicated that compound ZDLT-1 was a potent cytotoxic agent against HCT116 cells with cell apoptosis induction. Further, in silico prediction of physicochemical properties, drug-likeness, and ADME parameters suggested that compound ZDLT-1 is a promising anticancer agent. Taken together, the high potency cytotoxicity and class I HDACs inhibitory activity of compound ZDLT-1, which with the β-carboline 1,3,4-oxadiazole based hybrids as potent anticancer agents could be nominated for further modification and optimization.