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Hemorrhagic septicemia (HS) and mastitis caused by <i>Pasteurella (P.) multocida</i>, <i>Staphylococcus (S.) aureus</i> and <i>Streptococcus (Str.) agalactiae</i> are important ailments of the dairy industry especially in South Asia. The present study evaluates the efficacy of a locally prepared hemorrhagic septicemia (HS) and mastitis combined vaccine. To this end, a total of 70 HS, <i>S. aureus</i> and <i>Str. agalactiae</i>-free lactating (early stage of lactation) buffaloes (n = 45) and cows (n = 25), and 50 lactating (early stage of lactation) cows (n = 25) and buffaloes (n = 25) positive for <i>S. aureus</i>/<i>Str. agalactiae</i> were subjected to two doses of HS–mastitis combined vaccine with an interval of 21 days. Antibody response was monitored by ELISA up to six months (180 days). Antibody titers against HS and mastitis were significantly (<i>p</i> ˂ 0.05) higher in vaccinated groups as compared to the non-vaccinated groups. Cumulative mean somatic cell counts and mastitis severity scores in vaccinated groups were significantly lower (<i>p</i> < 0.05), and milk yield was significantly higher (<i>p</i> < 0.05) than those in the respective non-vaccinated controls. In conclusion, Montanide^®-adjuvanted HS–mastitis combined vaccine showed significant immunogenic effects in dairy cows and buffaloes. However, challenge studies remain overdue.