The application of plants with medicinal properties has been a fundamental practice in the history of medicine, aiming to treat and prevent diseases. The genus Dipteryx, Fabaceae family, comprises 14 species in South and Central America, standing out for its diversity of compounds, such as coumarins, flavonoids and terpenoids. The objective of this work with Dipteryx lacunifera from the Piauí state (Brazil) is investigating its chemical constituents and evaluating cytotoxic, antimicrobial and anticholinesterase potential. From the hexane extract of the leaves, five constituents were isolated: one steroid, β-sitosterol (1), two triterpenoids, lupeol (2) and betulin (3) and two sesquiterpenes, spathulenol (4) and (1aR*,2'R*,4aR*,7S*,7aS*,7bR*)-1,1,7-trimethyldecahydrospiro[cyclopropa[e]azulene-4,2'-oxiran]-7-ol (5). The compound 5 are reported for the first time as natural compound. By CG-MS, 19 additional compounds were identified, such as hydrocarbons, steroids, diterpenes and fatty acids. Constituent 1 and hexane fraction exhibited strong inhibition (>75%) against PC3 and HCT-116 tumor cell lines. Regarding anticholinesterase activity, the fraction demonstrated inhibition only at the highest concentration, while antimicrobial activity revealed efficacy against Gram-positive S. aureus and E. faecalis. This works presents, for the first time, a comprehensive approach to D. lacunifera leaves, highlighting their therapeutic potential and contributing to scientific knowledge of the biodiversity of the Piauí cerrado. A rare sesquiterpene was isolated for the first time as natural compound.