Organophosphorus pesticides are one of the most extensively used forms of pesticides that could adversely impact fertility in both animals and humans. The current research aimed to investigate the potential effects of long-time malathion administration on testosterone in serum, oxidative stress biomarkers in testicular tissue and sperm characteristics of rats as an experimental model. Twenty adult male Wistar rats were split into two groups: the control group (n = 10) and the malathion-treated group (n = 10) based on the medication received orally by gavage (3 times/week for 60 days). Long-time administration of malathion negatively affected testosterone level, sperm count, sperm viability, and sperm morphology when compared to the control group. Lipid peroxidation increased significantly in the malathion-treated group when compared to the control group. On the other hand, Malathion administration caused a significant reduction in testosterone level, activities of glutathione peroxidase, reduced glutathione, and superoxide dismutase enzymes in the testicular tissue of male rats. In conclusion, long-term with space interval administration of malathion had deleterious effects on testosterone level and testicular oxidative status as well as semen quality in male rats. Consequently, it is essential to monitor surveys of organophosphorus pesticide residues in plants to protect consumer health.