The objective of this study was to investigate the effect of whey protein concentrate (WPC) and selenium (Se) supplementation on sperm quality, antioxidant enzymes activity and lipid peroxidation in seminal plasma, liver and testis of roosters. Forty-five Ross-308 broiler breeder roosters aged 60 weeks were used for an eight-week period in a 3 × 3 factorial arrangement of dietary treatments. Three levels of WPC (0.0, 1.5 and 3.0% of diet) and selenium supplementation (0.0, 0.2 and 0.4 mg/kg of diet) with five replications were tested. Total and progressive sperm motility, sperm concentration, plasma membrane integrity, and sperm viability were significantly lower in birds treated with Se supplementation-free diet (P < 0.05). Also, abnormal sperms were significantly higher in Se supplementation-free diets when compared to the diets supplemented with 0.4 mg/kg Se (P < 0.05). The use of 1.5% of WPC resulted in significantly increased total and progressive sperm motility compared to the WPC-free diet (P < 0.05). Selenium at the level of 0.4 mg/kg along with 3.0% WPC were associated with significantly increased Glutathione peroxidase and superoxide dismutase activity in seminal plasma as compared to other levels (P < 0.05). The highest level of total antioxidant capacity (TAC) in seminal plasma was observed at the level of 0.2 mg Se (P < 0.05). Further, 3.0% WPC resulted in significantly increased TAC concentration in seminal plasma compared to the WPC-free diet (P < 0.05). Moreover, the Malondialdehyde (MDA) level of seminal plasma in selenium supplementation-free diets was significantly higher than those of other levels (P < 0.05). Glutathione peroxidase activity, TAC, and MDA levels in the testis and liver were not affected by the WPC and Se levels. It can be concluded that dietary inclusion of WPC and Se improved the semen quality, increased antioxidant enzymes activity and decreased lipid peroxidation in seminal plasma of  broiler breeder roosters.