The compounds, which are obtained from natural plants or microbes may offer potential as one of the strategies for the management of cholangiocarcinoma. Oblongifolin C (OC), a natural small molecule compound extracted and purified from Garcinia yunnanensis Hu, can activate the mitochondrial apoptotic pathway in human cervical cancer cells. However, the direct effects of OC on cholangiocarcinoma cells are not well defined. The effect of OC on cell apoptosis and its underlying mechanisms were investigated in cultured QBC939 cells by the methyl thiazol tetrazolium assay, mitochondrial membrane potential, ATP content and western blot analysis. The present study reported that the in vitro treatment of human cholangiocarcinoma QBC939 cells with different concentrations (5, 10, 20 and 40 µM) of OC decreased cell viability and induced apoptosis in a dose‑dependent manner. The results of the present study also showed that OC‑induced QBC939 cell apoptosis was mediated through the inhibition of autophagy and mitochondrial dysfunction (MtD). Additionally, inhibiting autophagy increased OC‑induced apoptosis and MtD, whereas exposure to the autophagy inducer, rapmycin, attenuated these changes. Together, the results of the present study are the first, to the best of our knowledge, to identify OC as a chemotherapeutic agent against human cholangiocarcinoma QBC939 cells in vitro via the regulation of autophagy and MtD.