The research aimed to investigate whether the combination of L-carnitine (LC) and thymoquinone (TQ) could lower the adverse effects of methotrexate (MTX), a widely used chemotherapy agent in cancer therapy, on testicular function. A total of seven groups were established (n=7); control group, LC (200 mg/kg PO), TQ (50 mg/kg PO), MTX (single IP dose of 20 mg/kg), LC+MTX, TQ+MTX, and LC+TQ+MTX. The use of MTX resulted in lower levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the blood, accompanied by a significant rise in testicular MDA levels. Additionally, there was a notable decline in GSH, CAT, and SOD, indicating reduced antioxidant activity in the testicles. Furthermore, the histopathological examination of the testicles revealed disruptions caused by MTX, and the expression of caspase-3 in the testicles was documented. The combination of LC and TQ with MTX significantly increased testosterone, FSH, LD levels and restored oxidative damage markers (p<0.05) and improved histopathological scores and caspase-3 expression. In conclusion, our results demonstrate that LC and TQ exhibit substantial protective effects against testicular toxicity induced by MTX, primarily attributed to their antioxidant properties.