In this study the effects of a new antibiotic combination, i.e., gentamycin, tylosin and linco-spectin (STLS) on post-thaw motion characteristics, plasma membrane integrity, sperm morphology and the total aerobic bacterial counts (TABC) in buffalo and Sahiwal bull semen were investigated. Ten ejaculates, five each from a buffalo and a Sahiwal bull, possessing more than 60% sperm motility were used. These ejaculates were diluted in Tris-citric acid (TCA) extender (at 37 °C; 50 X 106 spermatozoa/mi), containing either GTLS (gentamycin 500 g/ml, tylosin 100 g/ml and linco-spectin 300/600 g/ml), streptomycin 1000 g/ml and penicillin 1000 IU/ml (SP), or negative control with no antibiotics (NCON). Samples were cooled to 4°C in 2 hours, equilibrated at 4°C for 4 hours, filled in 0.5 ml straws, frozen in a controlled rate cell freezer and plunged into liquid nitrogen. Frozen semen was thawed at 37°C for 15 seconds. Post-thaw sperm motion characteristics, plasma membrane integrity and sperm morphology were determined. Total aerobic bacterial counts and the frequency of appearance of bacterial genera were determined in neat semen, after dilution, and after freezing and thawing. Mean motilities (visual; computer-assisted, linear and circular), velocities (straight-line, average path and curvilinear) and lateral head displacement (LHD) in post- thaw semen samples did not differ due to antibiotics or species. Same was true for sperm plasma membrane integrity. Morphologically abnormal spermatozoa were lower (P<0.05) in GTLS and SP than in NCON. Sperm cells possessing normal acrosomes were higher (P<0.01) in GTLS and SP than in NCON. Total aerobic bacterial counts in post-thaw samples were lower (P<0.05) in GTLS than in SP or NCON. Staphylococcus and micrococcus were lower in samples treated with GTLS than that of SP or NCON. Pseudomonas and E.coli were more frequent in buffaloes than Sahiwal bull samples. Proteus and corynebacteria were scarcely present. In conclusion, GTLS was not determintal to post thaw motion characteristics, sperm morphology and membrane integrity of buffalo and Sahiwal bull spermatozoa. Furthermore, it efficiently reduced the number of aerobic micro-organisms in buffalo and Sahiwal bull semen.