The aim of this study was to verify the existence of synergistic antibacterial effect between four essential oils (Aniba rosaeodora, Melaleuca alternifolia, Origanum vulgare, and Pelargonium graveolens) individually combined with the antibacterial drug Gentamicin. We investigated the effectiveness in vitro of the association of essential oil/Gentamicin, against fifteen different strains of Gram positive and Gram negative bacteria. The antibacterial effects of these oils in combination with Gentamicin were evaluated by using the MHB microdilution method, while gas chromatography (GC) and GC/Mass spectrometry were used to analyze the chemical composition of the oils. A synergistic interaction was observed against all tested strains with the associations between the essential oils Aniba rosaeodora/Gentamicin and Pelargonium graveolens/Gentamicin. In particular a very strong synergistic interaction was observed against Acinetobacter baumannii ATCC 19606 (FIC index = 0.11). In contrast, the essential oils Origanum vulgare and Melaleuca alternifolia in association with Gentamicin were less effective on bacterial species growth. In vitro interaction can improve the antimicrobial effectiveness of the Gentamicin and may contribute to reduce its dose correlated to side effects.