Introduction Praxelis genus comprises 24 species, however, only two species of this genus have been chemically investigated. Here we investigated Praxelis sanctopaulensis, a native plant from Brazil, that occurs mainly in Cerrado regions. Objective The goal was to identify the specialised metabolites from P. sanctopaulensis, and compare with those described from Praxelis and Chromolaena species. Methods The phytochemical study of P. sanctopaulensis was performed through different chromatography techniques, including high‐performance liquid chromatography (HPLC), gas chromatography flame ionisation detector (GC‐FID), and ultra‐high‐performance liquid chromatography high‐resolution tandem mass spectrometry (UHPLC‐HRMS/MS). The structures of the compounds were established based on spectroscopic analysis, total correlated spectroscopy (TOCSY), hydrogen decoupling and computational calculations was used to an unequivocal structural elucidation of a new sesquiterpene. The antioxidant activity was evaluated using 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), and antimicrobial assay was performed by the microdilution method. Comparison of the flavonoids described P. sanctopaulensis was carried out using principal component analysis. Results The phytochemical investigation of P. sanctopaulensis led to the isolation of a pair of diastereomers, praxilone A and praxilone B. Seven known compounds were isolated from this species, another 14 fatty acids were detected in hexane fraction, and 26 compounds were identified from ethyl acetate fraction. All these compounds are being described for the first time in this species, with the exception of viridifloric acid. The ethyl acetate fraction showed potent antioxidant activity. Conclusions Forty‐seven compounds are described from P. sanctopaulensis. The combination of different techniques of nuclear magnetic resonance (NMR) spectroscopy and computational calculations allowed the unequivocal structure elucidation of a new cadinene. The clustering analysis showed similarities between the flavonoids identified in P. sanctopaulensis and in Chromolaena species.