Acrocomia aculeata, known as macauba, is extensively distributed in the Southeast and Midwest of Brazil. Macauba fruit is used as food; however, its therapeutic properties are still poorly understood. The goal of the study was to investigate the chemical composition of macauba pulp oil (MPO) and anti-inflammatory, antimutagenic and antioxidant properties of MPO. The anti-inflammatory and antimutagenic properties were evaluated in Swiss mice treated with MPO per gavage or incorporated in the diet for 10 days. The serum of mice was subjected to lipid peroxidation evaluation and the in vitro MPO antioxidant activity was assessed. The organoleptic profile of MPO showed it was suitable for consumption and this composition showed unsaturated fatty acids with oleic acid the most abundant. The MPO presented anti-inflammatory properties, inducing a decrease in the migration of neutrophil and mononucleate cells to the peritoneal cavity of mice; it also inhibited the formation of micronuclei in mice. Furthermore, MPO presented antioxidant activity and high carotenoid content. Our data support the hypothesis that MPO has anti-inflammatory and antimutagenic properties, and these effects can correlate with fatty acid profile and antioxidant compounds. There is great potential for the application of MPO for nutritional purposes.