The present study aimed to study the effect of cryopreservation on the membrane, acrosome, DNA, and mitochondrial integrities of buffalo bull spermatozoa. Two adult buffalo bulls were enrolled in this study. Semen samples were collected once weekly from each bull over a period of six successive weeks by means of artificial vagina.  Samples were processed for cryopreservation. Individual motility, mitochondrial activity as well as membrane, acrosomal and DNA integrities of fresh and frozen-thawed spermatozoa were evaluated. Results showed that the procedures applied for freeze-thawing resulted in significant (P ≤ 0.001) reduction in sperm motility, sperm membrane, acrosome, mitochondrial and DNA integrities by 26.67, 29.17, 17.66, 40 and 4.11%, respectively. In conclusion, the process of freezing and thawing significantly reduced semen quality of buffalo bull in terms of various functional parameters including motility, membrane, acrosome, DNA and mitochondrial integrities.