The application of essential oils has been shown to be an effective and sustainable alternative against the multiplication of spoilage and pathogenic microorganisms in food. The aim of this work was to study the antimicrobial activity effect of Cerrado Rosemary (Baccharis dracunculifolia DC.), Pepper Rosemary (Lippia sidoides Cham.), Mandarin (Citrus nobilis Lour.), Mexican Lime (Citrus aurantifolia Swingle) essential oils, and the thymol and R-limonene compounds against strains of Penicillium expansum, Pichia kudriavzevii, Salmonella Thypimurium and Staphylococcus aureus. The essential oils were characterized in terms of their chemical composition, the Minimum Inhibitory Concentration, and Minimum Bactericidal and Fungicidal Concentration; using the microdilution method in 96-well plates. In addition, effects from mixtures of the essential oils, and the isolated compounds in sub-inhibitory concentrations were performed. The Pepper Rosemary essential oil and thymol, separately, showed the lowest Minimum Inhibitory Concentration values against all the microorganisms evaluated, with the lowest ranging from 0.03 to 1.25 mg. mL−1 for P. expansum and 0.31–1.25 mg. mL−1 for S. Thypimurium. Mandarin and Mexican lime, have an enhanced antimicrobial effect when used in mixture. The R-limonene compound showed a synergistic additive effect when combined with the essential oils, against S. aureus strains only. The application of thymol and Lippia was able to reduce growth by 3 log10 for S. aureus and S. Typhimurium in 24 h. Therefore, the Pepper Rosemary essential oil, applied freely or in combination with the thymol compound, has greater potential for use as an antifungal and bactericidal agent, against the microorganisms evaluated.