The present study aimed to investigate the cryoprotective effect of different combinations of glycerol (G), dimethyl sulphoxide (DMSO) and dimethylformamide (DMF) with chicken egg yolk (CEY) and duck egg yolk (DEY) on freezability of buffalo spermatozoa.  Semen samples were collected from 4 buffalo bulls and diluted with Tris-based extender supplemented with 7% (V/V) cryoprotectant including G, DMSO, DMF or a mixture of 3.5% G + 3.5% DMSO, 3.5% G + 3.5% DMF and 3.5% DMSO + 3.5% DMF. All the fore-mentioned aliquots were divided into two equal portions; one is supplemented with chicken's egg yolk (CEY) and the other with duck's egg yolk (DEY).  Post-thawing sperm motility, viability and acrosomal, plasmatic membrane and DNA integrities were assessed. The results clearly indicated that adding a mixture of 3.5% G + 3.5% DMSO with DEY to Tris extender significantly improved (p < 0.01) post-thawing sperm motility, viability index and percentage of spermatozoa with intact acrosomes, plasma membrane and DNA (58.33%±1.66%, 173.33±5.46, 69.00±1.58%, 65.00±1.15% and 96.91±0.13%, respectively) compared with the use of 7% G and CEY  (51.50±1.44%, 146.25±6.49, 61.00±2.30, 58.00±1.15% and 91.40±0.17, respectively).  In conclusion, motility and functional integrity of cryopreserved buffalo spermatozoa could be enhanced by using Tris extender containing a mixture of 3.5% glycerol + 3.5% DMSO with duck's egg yolk.