Five prenylflavonoids, 6‐prenylnaringenin (1), 8‐prenylnaringenin (2), 7‐O‐methyl‐8‐prenylnaringenin (3), 7‐O‐methyl‐6‐prenylnaringenin (4), and 4′‐O‐methyl‐6‐prenylnaringenin (5), were isolated from the traditional herb Mallotus conspurcatus Croizat (Euphorbiaceae). Compounds 1–5 revealed cytotoxic activity against cervical cancer (HeLa) cells with IC50 values ranging from 10.08 to 60.16 μm by MTT method, and interestingly, these prenylflavonoids were less toxic to normal HL‐7702 cells. Furthermore, compounds 1 and 5 could inhibit the c‐myc expression and telomerase activity and cause mitochondrial dysfunction. These findings might contribute to a better understanding of the biological activities of prenylflavonoids and lay the foundation for further studies on the cytotoxic activity of natural products isolated from M. conspurcatus.