Background This study was conducted for to explore the plausible pathway of Chenopodium album seed extract (CAE)-mediated sperm cell death. Study Design The role of CAE for its spermicidal action was assessed by (a) measuring lipid peroxidation, protein carbonyl content and intracellular glutathione content in CAE exposed sperm cells; (b) assaying antioxidant enzymes like catalase and superoxide dismutase (SOD); (c) analyzing protein expressions by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis; (d) fluorimetric measurement of intracellular H2O2 level and generation of reactive oxygen species (ROS) in CAE-treated sperm cells; and (e) DNA ladder formation study. Results CAE-induced sperm death is due to (a) lipid peroxidation of the sperm cell membrane, oxidation of some critical cellular proteins and depletion of intracellular reduced gluthathione, indicating production of ROS; (b) activation of Mn-SOD and inactivation of catalase favoring endogenous accumulation of H2O2; (c) generation of O2 − at an enhanced rate during oxidative stress as evidenced by increased Mn-SOD activity and protein expression; (d) accumulation of ROS in spermatozoa reflected in the fluorimetric experiments; and (e) increased production of O2 − and H2O2 induced apoptosis-like death in sperm cells as observed by DNA ladder formation. Conclusion The sperm death mediated by CAE is due to oxidative damage of cellular macromolecules by in situ generation of ROS.