The aim of this study was to evaluate the direct antibacterial activity, antibiotic modulation and toxicity of the essential oil of Ocimum gratissimum L. and its major compound Eugenol. The phytochemical analysis of the essential oil from leaves was investigated by Gas Chromatography coupled to Mass Spectrometry (CG-MS) and Eugenol was the major compound (63.25%). The Minimum Inhibitory Concentration and clinical antibiotics modulatory activities were performed using the 96-well plate microdilution methodology. Drosophila melanogaster was used as a model to assess the toxicity of the two substances. In conclusion, the eugenol and essential oil of Ocimum gratissimum L. showed direct antibacterial activity and associates with the tested antibiotics, which place them as promising agents against bacterial resistance. The essential oil showed less toxicity to fruit flies when compared to eugenol alone, although the results were numerically similar.