Aim: The study elucidates the impact of cryopreservation on lipid peroxidation (LPO), antioxidant potential, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm. Materials and Methods: Semen from eight mature cocks was pooled and diluted at 37 degrees C using a specific medium for red fowl species. The diluted semen samples were immediately refrigerated at 4 degrees C for 2 hours (0.275 degrees C min(-1)). Glycerol was then added to a final concentration of 20%, and the samples were equilibrated for 10 minutes at 5 degrees C before loading into 0.25 mL French straws. These straws were then frozen by placing them in liquid nitrogen vapor for 10 minutes and plunged into liquid nitrogen. Motility, viability, DNA integrity, antioxidant activity, and LPO were assessed before dilution (fresh semen), after equilibration (processed semen), and post-thawing (frozen semen). Results: Sperm motility, viability, DNA integrity, and mitochondrial activity decreased (p < 0.05) in processed and frozen semen compared with fresh semen. Nevertheless, the concentration of malondialdehyde (MDA) in sperm and seminal plasma was greater (p < 0.05) in frozen-thawed and processed semen compared with fresh semen. Multivariate regression analysis showed a negative impact of MDA concentration in sperm (R-2 = 0.90, Wilk's lambda = 0.003, p < 0.001) and seminal plasma (R-2 = 0.84, Wilk's lambda = 0.02, p < 0.001) on motility, viability, DNA integrity, and mitochondrial activity. Nonetheless, total antioxidant capacity (TAC) had a positive impact on sperm variables (R-2 = 0.82, Wilk's lambda = 0.096, p < 0.001). Conclusions: The decrease in motility, viability, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm was associated with an increase in LPO during cryopreservation. Furthermore, TAC was reduced during the freeze-thaw process, which was insufficient in protecting the sperm against high reactive oxygen species levels.