The present study analysed the mechanism of action of 2-Allyl-5-t-Butylhydroquinone purified from methanol extract of Chlorosarcinopsis eremi at different catalytic cycle of topoisomerase II enzyme. The result revealed that compound acted as topoisomerase II inhibitor and also as topoisomerase II poison. In vitro cytotoxicity assay was also performed using Breast cancer cell line (MCF 7 cell lines). Cell viability was measured using the MTT assay, and % cell inhibition was calculated. The Half Maximal Inhibitory Concentration (IC50 value) of 2-Allyl-5-t-Butylhydroquinone on MCF7 cell lines indicated moderate cytotoxic activity against MCF7 cells. The study suggests that 2-Allyl-5-t-Butylhydroquinone has potential applications in the development of novel anticancer therapeutics.