Unonopsis guatterioides (A.DC.) R.E.Fr., is found mainly in the Pantanal, Cerrado and Amazon biomes and, some species of this genus are used in folk medicine. The analysis by HPLC-ESI-MS revealed the presence of alkaloids previously reported for Unonopsis genus, such as asimilobine, anonaine, nornuciferine, glaucine and norglaucine. In contrast, the heliamine, norjuziphine and anomuricine alkaloids are being reported for the first time in the Unonopsis genus, while this is the first report of azafluoranthene alkaloid triclisine in the Annonaceae family. These results showed the promising application of mass spectrometric monitoring of complex extracts in the search for novel natural products for the food, pharmaceutical, and cosmetic industries, thus simplifying phytochemical analysis. Bioactive analysis based on antioxidant activity indicated that ethanolic extracts of the peels and pulps of the fruits from U. guatterioides showed low scavenging activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with IC50 values > 100 mu g*mL(-1), using as reference the ascorbic acid (IC50 < 50 mu g*mL(-1)). These results are in concordance with the chemical profiles, whose major compounds proved to be O-substituted phenolic derivatives. Furthermore, ethanolic extracts of the peels (UGP-1) and pulps (UGP-2) of the fruits from U. guatterioides showed weak activity against Staphylococcus pseudintermedius, S. aureus and S. epidermidis, with MIC values above 1000 mu g*mL(-1). The combination of the ethanolic extract of the pulps of fruits from U. guatterioides and ampicillin resulted in an additive effect (FICI = 1.0), when tested against S. aureus and a strain of S. epidermidis. These results suggest that the pulps' ethanolic extract, when combined with the antibiotic ampicillin, can strengthen the therapy for S. aureus and S. epidermidis infection. Additionally, no genotoxic activity of the ethanolic extracts of fruits from U. guatterioides (UGP-1 and UGP-2) was detected at the tested concentrations (0.25, 1.25, 2.5 and 5.0 mg*mL(-1)). The genotoxic property was performed using the Somatic Mutation and Recombination assay (SMART Test), in vivo, in somatic cells of Drosophila melanogaster. (c) 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.