Display omitted •A novel series of nine quinoxalinyl-hydrazones was synthesized and characterized.•(E)-2-2-(2-Pyridin-2-ylmethylene)hydrazinylquinoxaline (PJOV56) showed strong cytotoxic activity against a panel of human tumor-derived cancer cell lines.•PJOV56 exhibited antiproliferative activity against HCT116 cells suppressing cell growth in the time- and dose-dependent manner.•Low concentrations (1.5 and 3.0 µmol.L−1) of PJOV56 induced cell cycle arrest in S-phase without apparent cell death in HCT116 cells.•High concentration (6.0 µmol.L−1) of PJOV56 led to a cell cycle arrest in G0/G1-phase and to a significant level of apoptotic cell death in HCT116 cells.•PJOV56 induced intense vacuolization in HCT116 cells that ressemble autophagic process induction. Quinoxaline derivatives are reported as antineoplastic agents against a variety of human cancer cell lines, with some compounds being submitted to clinical trials. In this work, we report the synthesis, characterization and cytotoxicity potential of a new series of quinoxalinyl-hydrazones. The most cytotoxic compound was (E)-2-2-(2-pyridin-2-ylmethylene)hydrazinylquinoxaline (PJOV56) that presented a time-dependent effect against HCT-116 cells. After 48 h of incubation, PJOV56 was able to induce autophagy and apoptosis of HCT-116 cells, mediated by upregulation of Beclin 1, upregulation of LC3A/B II and activation of caspase 7. Apoptosis was induced along with G0/G1 cell cycle arrest at the highest concentration of PJOV56 (6.0 µM). Thus, PJOV56 showed a dose-dependent mode of action related to induction of autophagy and apoptosis in HCT-116 cells.