Essential oils are natural substances composed of terpenoids and phenylpropanoid molecules that have many biological activities. Because of their activity, essential oils are widely used in foods, cosmetics, and pharmaceutical products, but new applications of such mixtures are still dependent on separation processes that are able to produce compounds with specific standardized and reproducible compositions. This work studies the fractionation of lemongrass essential oil by vacuum distillation with the goal of obtaining an antimicrobial fraction that acts against Salmonella choleraesuis, Pseudomonas aeruginosa and Staphylococcus aureus. The samples were analyzed by gas chromatography–mass spectrometry and evaluated for antimicrobial activity using the indirect bioautography method and minimum inhibitory concentrations. Vacuum distillation was found to be an efficient method for obtaining distinct fractions with increased antimicrobial activity. The fractions that showed the best response against the three microorganisms tested were F3b, F3a1, F2, F3a2 and F1, with the most effective being F3a2, which also presented the highest yield.