The species Hancornia speciosa (mangabeira) has varied potential. The bark has astringent properties, latex is used for tuberculosis, ulcers, herpes, dermatoses and warts, leaf tea is used for menstrual cramps and the root decoction to treat dislocations and hypertension. The aim of this work was to analyze the chemical composition of the fixed oil and antibacterial activities alone or in association with aminoglycosides against standard and MDR bacteria using broth microdilution assays. In the analysis of the oil by GC/MS, a high content of unsaturated fatty acids (73.46%) was identified in relation to saturated fatty acids (26.15%). Palmitic acid (22.49%) and elaidic acid (69.50%) were the main fatty acids identified. The antibacterial test results showed a more significant oil activity against Staphylococcus aureus SA–ATCC 6538 (MIC = 512 μg/mL). For other strains including standards and multidrug resistant bacteria, the oil presented MIC ≥ 1024 μg/mL. In association with antibiotics, the oil was able to improve antibacterial activity against bacterial strains. A synergic effect was observed for S. aureus SA–10, with MIC reduction of amikacin and gentamicin by 50.00 and 60.00%, respectively. The most significant association was found for Escherichia coli EC–06, with a reduction in MIC of 81.25%. The results indicate that seed oil of H. speciosa has the potential to act on bacterial resistance to aminoglycoside antibiotics.