Abstract: In order to investigate the effects of tiamulin, neomycin, tetracycline, fluorophenicol, penicillin G, Linco-Spectin (0.15 mg mL-1 lincomycin + 0.3 mg mL-1 spectinomycin), erythromycin and oxytetracycline on controlling bacterial contaminations of the river buffalo semen, 120 mL diluted buffalo bull semen (diluted by tris-egg yolk extender) was divided into 5 mL tubes after initial evaluation and before (control sample) and at 0, 2, 6, 12 and 24 h after adding each of the above antibiotics at the recommended dose (D) and twice the recommended dose (Dx2) to the semen samples, each sample was cultured 4 times on Muller-Hinton agar medium and the results were recorded after 18 h incubation at 37°C. Tiamulin, tetracycline, neomycin and fluorophenicol were ineffective. Oxytetracycline was effective in both D and Dx2 (p<0.001). Penicillin G in both D and Dx2 was effective (p<0.001). Linco-Spectin was effective, though not significant, in D at 2 h and in Dx2 at 0 h only. Erythromycin in D was not significantly effective, but, in Dx2 was effective (p<0.001). Duration of the antibiotic exposure had no significant effect on the antibiotic potentials except for Linco-Spectin at 2 h (p<0.014). The biochemical tests identified the contaminant bacteria as being a member of Arcanobacter (Corynebacterium) sp. In the next step, the semen sample of the same bull was taken, semen quality tests were carried out and the semen was diluted with the same extender (tris-egg yolk) + 7% glycerol, containing a double dose (Dx2) of these antibiotics and semen quality tests were carried out immediately after dilution, 18 h after storage at 4°C and after the semen was packed in the straws, frozen in liquid nitrogen (-196°C) and later thawed in 37°C water bath to investigate whether these antibiotics have any adverse effect on the spermatozoa during the process of freezing and thawing. The comparison of the results with those of the control group (the sample undergone the same process without adding antibiotics) indicated that oxytetracycline adversely affected sperm motility at 0 and 18 h, all the antibiotics had a lower percentage of sperm abnormal morphology than the control at 0 and 18 h, except for Linco-Spectin at 18 h and after freezing-thawing and tetracycline after freezing and thawing the sample which were the same as the control. Sperm viability was not affected by antibiotics before and after freezing. It was concluded that oxytetracycline and penicillin G in both D and Dx2 were effective in controlling seminal bacterial contaminations and because of the adverse effect of oxytetracycline on the sperm motility and morphology, it proved not to be suitable for this purpose but penicillin G could be recommended as an additive to the semen extenders.