This study evaluated the in vitro activity of essential oil extracted from the leaves of Myracrodruon urundeuva. The oil was obtained by hydro-distillation and characterized by Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Gas Chromatography with Flame Ionization Detector (GC-FID). The antibacterial activity was evaluated by the broth microdilution technique and the MIF was determined by using growth indicator CTT (2,3,5-triphenyl-tetrazolium) and CBM in BHI agar. The oil’s cytotoxicity was evaluated in HeLa, HEK-293, and Vero E6 cells using MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium. The oil showed chemical markers, including α-pinene (87.85%), trans-caryophyllene (1.57%), limonene (1.49%) and β -pinene (1.42%), and activity against all strains: Staphylococcus aureus (MIC = MBC = 0.22 mg/mL), Staphylococcus epidermidis (MIC = 0.11 mg/mL and MBC = 0.22 mg/mL), Escherichia coli (MIC = 0.88 mg/mL and MBC = 1.75 mg/mL), Pseudomonas aeruginosa (MIC = MBC = 7 mg/mL) and Salmonella Enteritidis (MIC = MBC = 0.44 mg/mL). In vitro cytotoxicity tests showed that the oil is not toxic and has slight antitumor activity. We conclude that the M. urundeuva oil results are promising, with prospects of being pharmacologically viable.