Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based semen extender on enzymatic and non-enzymatic antioxidant activity in equine semen cryopreservation. Fifteen ejaculates from six fertile Criollo stallions were cryopreserved using a commercial citrate-Hepes, egg yolk, skim milk and glycerol extender. Activities of catalase, glutathione peroxidase and superoxide dismutase and total radical-trapping antioxidant potential were assessed in raw semen, semen diluted in extender and thawed semen. All three enzymes showed higher activities in raw semen than in diluted or in thawed semen (P < 0.01), but enzyme activities did not differ significantly between diluted and thawed semen samples (P > 0.05). Non-enzymatic antioxidant defenses did not differ among any of the stages in the cryopreservation process (P > 0.05). In conclusion, the present study shows that dilution of semen with skim milk-egg yolk based extender after centrifugation compensates for the non-enzymatic antioxidant protection (but not enzymatic antioxidant defense) lost with seminal plasma removal. The absence of correlation between seminal and antioxidant parameters suggests that the compensation was enough for semen protection against oxidative stress, or antioxidant protection plays a minor role on semen from fertile stallions.