The aim of this study was to examine the effects of different antibiotics administered intramuscularly with a different administration times on the humoral immune response resulted from intraocular vaccination with attenuated B. melitensis Rev.1 vaccine in sheep. The results showed a significant difference in the effect of different antibiotics used with a significant difference according to the administration time between 0 time, 7 and 14 days from vaccination. Oxytetracycline and amoxicillin significantly inhibit the humoral immune response when administrated at 0, 7 and 14 days of Brucella vaccination comparing with groups treated with sulphadimidine, penicillin-dihydrostreptomycin and the non-vaccinated groups while sulphamethazine – trimethoprim inhibit the humoral immune response when administrated at 0 time of vaccination comparing with groups treated with sulphadimidine, penicillin-dihydrostreptomycin and the non-vaccinated groups. The results indicate that the failure of Brucella vaccination could happen when antibiotics administered within the first 14 days after vaccination.