Varicocele has been associated with decrease in seminal parameters. Selenium (Se), copper (Cu), and zinc (Zn) are trace elements essential for normal spermatogenesis of mammals and play a critical role as antioxidant defense system enzymes. Se, Cu, and Zn are associated with sperm quality in fertile and infertile men. However, there is little information about Se, Cu, and Zn concentrations in semen in patients with varicocele and its association with seminal parameters. The purpose of this study was to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. Total Reflection X-Ray Fluorescence was used for the fist time in the seminal fluid analysis. The concentration of selenium in men with varicocele was smaller than the normozoospermic group, while no differences were observed for both concentrations of zinc and copper. A significant positive correlation between zinc and selenium concentration was observed. Selenium in seminal plasma correlates with a good spermatozoa concentrations, motility, and morphology. Additionally, a significant positive correlation was observed between zinc levels and sperm count. In conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters. A study should be conducted to evaluate the benefits of both zinc and selenium supplementation to improve seminal parameters in patients with varicocele.