Every year, more than 500,000 new cases of cervical cancer are reported, making it the fourth leading cause of cancer globally. Although human papillomavirus (HPV) vaccines show promise as a protective measure, HPV-related cancers remain a public health problem since the vaccines, which are only specific to certain viral types, are unavailable for mass distribution. Furthermore, the effects of toxicity following ionizing radiation therapy have reoriented views toward the search for radiosensitizers that can reduce toxicity as a consequence of decreased radiation doses. Here, we isolated ergosterol peroxide (EP) from Pleurotus ostreatus and purified it to test its potential effects in vitro. We thus observed that a gradual increase in EP dose correlates with a loss of viability in HeLa and CaSki cervical cell lines. Dose/response curves were constructed using cervical cancer cell lines, as well as normal human peripheral blood mononuclear cells. The selectivity of EP in human lymphocytes and cervical cancer cell lines was tested, and no toxicity was found in normal cells. A combination of treatments revealed a radiosensitizer effect in HeLa cells, when measuring the exposure to EP followed by irradiation with 137Cs. Our findings suggest that EP may be effective as a radiosensitizer in treating cervical cancer.