Background: Tumor cells require proficient autophagy to meet high metabolic demands and resist chemotherapy, which suggests that reducing autophagic flux might be an attractive route for cancer therapy. However, this theory in clinical cancer research remains controversial due to the limited number of drugs that specifically inhibit autophagy-related (ATG) proteins. Methods: We screened FDA-approved drugs using a novel platform that integrates computational docking and simulations as well as biochemical and cellular reporter assays to identify potential drugs that inhibit autophagy-required cysteine proteases of the ATG4 family. The effects of ATG4 inhibitors on autophagy and tumor suppression were examined using cell culture and a tumor xenograft mouse model. Results: Tioconazole was found to inhibit activities of ATG4A and ATG4B with an IC50 of 1.3 µM and 1.8 µM, respectively. Further studies based on docking and molecular dynamics (MD) simulations supported that tioconazole can stably occupy the active site of ATG4 in its open form and transiently interact with the allosteric regulation site in LC3, which explained the experimentally observed obstruction of substrate binding and reduced autophagic flux in cells in the presence of tioconazole. Moreover, tioconazole diminished tumor cell viability and sensitized cancer cells to autophagy-inducing conditions, including starvation and treatment with chemotherapeutic agents. Conclusion: Tioconazole inhibited ATG4 and autophagy to enhance chemotherapeutic drug-induced cytotoxicity in cancer cell culture and tumor xenografts. These results suggest that the antifungal drug tioconazole might be repositioned as an anticancer drug or chemosensitizer.