Previous research has shown benefits of dietary fish oil supplementation on semen quality of boars. However, little is known about how antioxidant protects lipid peroxidation on spermatozoa from n-3 polyunsaturated fatty acid (PUFA) addition. This study evaluated the effect of oregano essential oil (OEO) supplementation on semen antioxidant status and semen quality in boars fed a diet enriched with fish oil. Thirty-four mature boars of proven fertility, received daily 2.5 kg basal diet top-dressed with 45 g soybean oil and 15 g fish oil to meet the n-3 PUFA requirement of spermatozoa, randomly allocated to one of four groups supplemented with 100 mg α-tocopheryl acetate kg−1 (control), or 250 or 500 or 750 mg OEO kg−1 for 16 weeks. Semen was collected at weeks 0, 8, 12 and 16 for measurements of sperm production, motion characteristics, sperm α-tocopherol content, antioxidant enzyme activities, reactive oxygen species (ROS), DNA damage (8-hydroxydeoxyguanosine, 8-OHdG), lipoperoxidation (malondialdehyde, MDA) and seminal total antioxidant capacity (TAC). Sperm production and motion characteristics were similar (p > .05) among groups throughout the experimental week 16, but increased (p < .01) with experimental week. Although higher α-tocopherol content and superoxide dismutase (SOD) activities were in OEO group spermatozoa, feeding diet with 500 mg/kg OEO resulted in elevation in seminal TAC, decrease in sperm ROS, MDA and 8-OHdG than control group (p < .05). Overall, these results support the view that oregano essential oil has a positive effect on antioxidant capacity in boar when used fish oil.