The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P amp;lt; 0.05) higher total motility (MOT) (73.78 +/- 2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 +/- 0.38, 16.78 +/- 0.38) in comparison with the control group (MOT: 58.48 +/- 2.92; PROG: 9.08 +/- 0.38). Sperm viability (Vi) was higher (P amp;lt; 0.05) in n1t3 (74.62 +/- 1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 +/- 1.91, 63.73 +/- 1.91, respectively) compared to the control groups (Vi: 66.85 +/- 1.55; Mi: 53.18 +/- 1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 +/- 0.31). There was no significant difference for SOD and CAT values with the use of various extenders. In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.