Low fertility in buffaloes with frozen-thawed semen is attributed to sperm damage that is related to formation of ice crystals during freeze/thaw process. This study was conducted to evaluate the freezability of buffalo bull semen extended with four types of extenders, including citrate egg-yolk (CEY, control), CEY with ethanolic Moringa Olifera Leaf Extract (eMOLE) as replacement of antibiotics (CEYm), eMOLE and eMOLE with conventional antibiotic (eMOLEa) with or without Coenzyme Q10 as antioxidant. Semen was subjected to calculate progressive motility, livability, abnormality, acrosome status and integrity of sperm membrane in semen after extending, equilibrating and thawing processes. Four sexually mature buffalo bulls (4-5 y and 400-450 kg LBW) were used for semen collection, and semen was collected twice weekly for 4 successive weeks. Semen ejaculates of ≥70% as mass motility percentage were pooled and extended at 37°C by the four types of extenders without or with 30 µM Co-Q10). Results cleared that progressive motility, livability, abnormality, acrosomal damage and plasma membrane integrity percentages improved (P<0.05) in buffalo semen after dilution, equilibration and thawing, while activity of AST, ALT and LDH in seminal plasma of post-thawed semen reduced (P<0.05) by extension of semen with eMOLE as a new extender. All sperm parameters and enzyme activity were maintained by replacing antibiotics in CEY extender with 4 ml eMOLE. All sperm parameters were improved (P<0.05) and enzyme activity was maintained by adding 30 µM of coQ10 as compared to free extenders. Fertility rate of buffalo cows inseminated with semen cryopreserved in extender (eMOLE+coQ10) was the highest (91.6%), followed by eMOLE without coQ10 (83.3%) and the lowest for control CEY (66.6%). In conclusion, eMOLE as a new extender, is promising for the extension and preservation of buffalo semen. Also, adding 4 ml of eMOLE could be a good substitute for the antibiotic component of CEY as a conventional semen extender. Moreover, supplementing semen extenders with antioxidant addition (30 µM CoQ10) is important to improve function of spermatozoa and sperm fertility of cryopreserved semen of buffaloes.