This study aimed to investigate the effect of dietary n-3 fatty acid and ginger (Zingiber officinale) supplementation on semen quality, sperm fatty acids, and reproductive performance of roosters. Seventy-two roosters (30 weeks old) were randomly allocated into 4 dietary treatments including 1) basal diet as the control group (F0G0), 2) basal diet supplemented with 20 g/kg fish oil (F2G0), 3) basal diet supplemented with 30 g/kg ginger powder (F0G3), and 4) basal diet supplemented with 20 g/kg fish oil and 30 g/kg ginger powder (F2G3) for 10 consecutive weeks. The levels of sperm linolenic, EPA, and DHA were higher in fish oil-fed roosters (P<0.05). A lower percentage of sperm linoleic and arachidonic acids were recorded in F2G0 and F2G3 groups (P<0.05). At 38 weeks of age, a higher sperm volume was found in F0G3 roosters than in the F0G0 and F2G0 groups. From week 34 to the end of the experiment, sperm viability, sperm abnormality, and sperm motility were significantly improved in the GP-fed roosters (F0G3 and F2G3 groups) (P<0.05). Significantly lowest sperm concentration overall the experiment was observed in the F2G0 group (P<0.05). Higher testosterone levels and lower malondialdehyde (MDA) content were recorded in F0G3 and F2G3 groups in comparison to the F2G0 (P<0.05). Also, the fertility rate of collected eggs from F0G3 and F2G3 groups was higher compared to F2G0 group (P<0.05). In conclusion, although the use of fish oil in roosters’ diets alone had a negative effect on some parameters related to reproductive performance, the use of ginger powder alone or along with fish oil improved semen quality and fertility potential.