Owing to the economic and zoonotic importance of Brucella melitensis, the present study aimed to evaluate inactivated Brucella melitensis S-16M vaccine prepared by using formalin 0.5% then mixed with montanide ISA-206 oil in a ratio of (1:1) as a protective vaccine for control of brucellosis in goats and sheep. Adult Brucella free guinea pigs (350-500g) were divided into four groups and the prepared vaccine was evaluated for protection of guinea pigs against challenge infection with virulent B. melitensis S-16M in comparison to Rev-1 vaccine and to measure the humeral and cellular immune response of the guinea pigs by using different serological tests such as Rose Bengal Plate Test (RBPT), Buffered Acidified plate test (BAPA) and ELISA. Moreover, it was evaluated in susceptible sexually mature goats by using different serological tests such as RBPT, BAPA, Standard tube agglutination and Complement fixation tests to assure that it could be used safely in the field. It was found that the protective activity of the prepared vaccine was 2.944 while that of Rev-1 was 1.66 in guinea pigs. Differences between the protection afforded by the oil adjuvant vaccine and standard Rev1 vaccine were not significant (at P ≥ 0.05) but the prepared one was potent, safer and stimulated humeral and cellular immunity. In comparison with PBS inoculated (unvaccinated) group, the protection given by the prepared vaccine was significant and also it was safe and immunogenic as antibody titer in sera of vaccinated guinea pigs and goats was monitored with serological tests.