Abstract: Poultries are infected to different kinds of microbial infections during their growth. For prevent of these diseases, many farmers use the synthetic antimicrobial agents. Whereas, the poultries participate in food cycle of human, the residues of these agents enter in human and cause many undesired side effects. In this study, the antimicrobial activity of Respitol-B and its main components (eucalyptus oil and menthol) was evaluated on different kinds of microorganisms including gram positive, gram negative bacteria, yeast and fungi in vitro conditions by disc diffusion and micro broth dilution assays. The gram positive bacteria, yeast and fungi is more sensitive than Gram negative ones to Respitol-B. Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium is less sensitive to Respitol-B. Evaluation of menthol and eucalyptus oil for their antimicrobial activities exhibited that the antimicrobial activity of menthol is higher than that of eucalyptus oil. Eucalyptus oil had the best effect on Vibrio cholerae, Staphylococcus aureus, Aspergillus flavus but had no effect on others. The antimicrobial activity of menthol is observable and its presence in Respitol-B enhances the antimicrobial activity of Respitol-B. Respitol-B as a 100% herbal drug has antimicrobial effect and can be used as alternative therapy for preventing and controlling of infections.