Objective:To evaluate the in vitro antimicrobial activity of the Eucalyptus globulus essential oil, and of the xylitol and papain substances against the following microorganisms: Pseudomonas aeruginosa; Samonella sp.; Staphylococus aureus; Proteus vulgaris; Escherichia coli and Candida albicans. Method: The in vitro antimicrobial evaluation was used by means of the agar diffusion test and evaluation of the inhibition zone diameter of the tested substances. Chlorhexidine 0.5% was used as control. Results: The Eucalyptus globulus oil showed higher inhibition than chlorhexidine when applied to Staphylococcus aureus, and equal inhibition when applied to the following microorganisms: Escherichia coli, Proteus vulgaris and Candida albicans. Papain 10% showed lower antimicrobial effect than chlorhexidine in relation to Candida albicans. Xylitol showed no inhibition of the tested microorganisms. Conclusion: The Eucalyptus globulus oil has antimicrobial activity against different microorganisms and appears to be a viable alternative as germicidal agent hence, further investigation is recommended.