Cryopreservation of Asian elephant semen remains problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. The present study compared the effects of the penetrating cryoprotectants glycerol and dimethyl sulfoxide (DMSO) in the freezing extender, in conjunction with the surfactant Equex STM paste, on Asian elephant spermatozoa during freezing and thawing. Eight ejaculates from three mature bulls were used for this study. Semen was diluted with Tris egg yolk-based extender which contained either glycerol or DMSO, either with or without Equex, and frozen over liquid nitrogen in straws. Sperm motility, membrane, acrosome, and DNA parameters were assessed over 2 h incubation post-thaw to evaluate the cryoprotective capabilities of each freezing extender. Spermatozoa frozen with glycerol resulted in higher total (P < 0.001) and progressive (P < 0.05) motility compared to spermatozoa diluted with DMSO, regardless of Equex inclusion in the freezing extender. When in the presence of Equex, Asian elephant spermatozoa demonstrated higher proportions of viability (P < 0.001) and intact acrosomes (P < 0.001) post-thaw, regardless of the type of cryoprotectant. A large variance between individual bulls was noticed when assessing sperm DNA integrity, nevertheless freezing extender with glycerol plus Equex showed the highest proportion of spermatozoa with intact DNA after 2 h post-thaw (P < 0.05). Altogether, these results indicate a Tris egg yolk-based freezing extender containing cryoprotectants glycerol and Equex should be integrated into current Asian elephant semen cryopreservation protocols to yield the best sperm quality post-thaw and thus improve fertilising potential.