Resveratrol is a natural grape-derived polyphenol with potent antioxidant properties that protect spermatozoa against lipid peroxidation (LPO) by eradicating free radicals. The objectives of this study were to 1) appraise the effects of resveratrol in extender on post-thaw quality parameters, antioxidant enzymes, adenosine triphosphate (ATP), DNA fragmentation, LPO and 2) fertilizing capability of buffalo bull spermatozoa. Semen was collected from four fertility proven bulls with artificial vagina thrice, evaluated initially, and diluted in five different extenders containing resveratrol (T4 = 100 μM, T3 = 50 μM, T2 = 20 μM, T1 = 10 μM), and control (no resveratrol), and evaluated after post-dilution and post-thawing stage of cryopreservation. Analysis of variance revealed that, there was no difference (P > 0.05) in any parameters due to treatments at post-dilution. However, at post-thawing, sperm progressive motility (%), plasma membrane integrity (%), mitochondrial membrane potential (%) and ATP levels (nmol/106) were found higher in semen samples cryopreserved in T3 and 4 than control. Sperm supravital plasma membrane integrity (%) and viable/acrosome integrity were found higher in semen samples cryopreserved in T4 than control and T1. Furthermore, sperm catalase (U/mL), glutathione peroxidase (μM) and superoxide dismutase (U/mL) concentrations were found significantly higher in resveratrol treated groups as compared to the control. Conversely, DNA fragmentation (%) and LPO (μM/mL) were significantly (P > 0.05) decreased in semen samples cryopreserved in T4 in comparison to the control. Fertilizing capability was found higher in semen samples cryopreserved in T4 as compared to the control (%, 77.33 vs. 57.41, P < 0.05). It is concluded that the addition of resveratrol in extender ameliorates quality parameters, antioxidant enzymes levels and fertilizing capability, and alleviate DNA fragmentation and LPO in buffalo spermatozoa during cryopreservation.