The aim of the study was to evaluate the antimicrobial activity of essential oils in vitro for possible application to reduce the content of microorganisms in the air of animal houses. The essential oils of Cymbopogon citrarus L. and Malaleuca alternifolia L. were screened against bacteria Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and yeast Candida albicans. The minimal inhibitory concentration of the active essential oils was tested using broth dilution assay. The essential oils concentrations ranged from 0.1-50.0%. The combined effects of essential oils were tested for Malaleuca alternifolia L. and Cymbopogon citrarus L. concentrations ranged from 0.005-50.0%. The oils showed a wide spectrum of antibacterial activity. Concentrations of 0.1-0.5% of Cymbopogon citrarus L. and Malaleuca alternifolia L. reduced total microorganisms count of Proteus mirabilis and Candida albicans. High antibacterial activity was also revealed for Cymbopogon citrarus L. with bactericidal concentrations of 0.8% for Escherichia coli, 5.0% for Enterococcus faecium, 5.0% for Pseudomonas aeruginosa and 8.0% for Staphylococcus aureus. Bactericidal concentrations of Malaleuca alternifolia L. were 5.0% for Pseudomonas aeruginosa and Enterococcus faecium, and 8.0% for Staphylococcus aureus. The essential oils of Cymbopogon citrarus and Malaleuca alternifolia may be a promising alternative of air disinfection in animal houses.