Previously undefined genotoxic and antigenotoxic potentials of hesperetin were elaborated in Drosophila melanogaster, upon inducing the DNA damage with ethyl methanesulfonate (EMS), proven alkylating agent and mutagen, within somatic cells. Upon the EMS-mediated in vivo alkylation, the O⁶-ethylguanine and O⁴-ethylthymine lesions emerged leading to the aberrant G=T and T=G pairing. Being applied in the equimolar concentration related to the alkylating agent, hesperetin significantly reduced the EMS-influenced DNA damage, as verified by the comet assay, implying that it has acted as a powerful Drosophila melanogaster’s Topoisomerase IIα (dmTopIIα) inhibitor. The dmTopIIα inhibition has been confirmed employing the electrophoresis on D. melanogaster plasmid DNA (dmPDNA) relaxation level, enzymatic and fluorescence assaying on dmTopIIα’s ATPase level and DNA-Binding and Cleavage Region, respectively, and molecular docking. Thus, as an antigenotoxic agent, hesperetin exerted dual pharmacology: within the dmTopIIα hesperetin acted as an ATPase uncompetitive inhibitor (as confirmed by spectrophotometric studies), denying the dmTopIIα energy for enzyme-catalized cleavage of double-strand containing the G=T and T=G pairings; within the dmTopIIα DNA-Binding and Cleavage Region hesperetin exerted no intercalating features (as verified by fluorescence quenching) but instead and blocked the EMS approach to either guanine and thymine, prevented the alkylation, and consequent dmTyr803-catalysed cleavage of normal double-strand (as certified by molecular docking). Conclusively, hesperetin could be used as a supplement for alkylating agent-based cancer therapy in terms of preventing the alkylation agent to cause unnatural lesions and aberrant pairing.