The abiu-do-mato (Pouteria caimito) is a fruit highly valued by Amazonian populations; however, its chemical composition and properties remain largely unexplored. This study aimed to fill this gap by analyzing the fruit's chemical profile and evaluating its antioxidant and cytotoxic properties. The research assessed proximate composition through physicochemical analysis, mineral content via atomic absorption spectrophotometry, and antioxidant activity using DPPH, ABTS, and FRAP assays. Total phenolic content was determined with the Folin-Ciocalteu reagent, while fatty acid and volatile compound profiles were identified using GC-MS. Additionally, NMR and HRMS were employed to characterize the chemical composition of the pulp and peel, and cytotoxicity was evaluated in vitro. Results showed that the peel contained the highest levels of antioxidant activity and phenolic compounds, followed by the pulp and seeds. GC-MS analysis of hexane extracts identified 19 compounds, primarily volatiles and fatty acids, with palmitic, elaidic, and linoleic acids being the most abundant. Methanolic extracts revealed 17 compounds, including catechin and its derivatives. Cytotoxicity assays indicated no toxic effects on the tested cells. These findings enhance the understanding of Pouteria caimito, highlighting its nutritional potential and possible applications in food and health-related industries.