The aim of this work was to evaluate the antibacterial activity of Copaifera duckei oleoresin and to determine its possible mechanism of action against bacteria of clinical and food interest. The antibacterial activity was determined by agar diffusion and dilution methods; the mechanism of action by transmission electron microscopy and by SDS-PAGE; the bioactive compounds by bioautography; and the chemical analysis by GC/MS. Oleoresin showed activity against nine of the 11 strains of bacteria tested. Bacillus cereus was the most sensitive, with a MIC corresponding to 0.03125 mg ml−1 and with a bactericidal action. Oleoresin acted on the bacterial cell wall, removing proteins and the S-layer, and interfering with the cell-division process. This activity probably can be attributed to the action of terpenic compounds, among them the bisabolene compound. Gram-negative bacteria tested were not inhibited. C. duckei oleoresin is a potential antibacterial, suggesting that this oil could be used as a therapeutic alternative, mainly against B. cereus.