BACKGROUND: Pot-pollen is a fermented product stored by stingless bees in cerumen pots and traditionally used as food or medicine by natives in tropical regions. Knowledge of pot-pollen composition from the Amazon region is important to strengthen the breeding of native bees and consequently contribute to sustainable development in this region. The aim of the present study was to determine the chemical composition of Melipona seminigra pot-pollen from Amazonas, Brazil. RESULTS: We report the identification of 21 phenolic compounds using ultra-performance liquid chromatography– electrospray ionization–quadrupole time-of-flight–mass spectrometry. Overall, the predominant constituents of pot-pollen were protein (333.8–470.7 g kg⁻¹) and dietary fibers (247.6–353.3 g kg⁻¹). The predominant fatty acids were polyunsaturated (44.95–59.57% of total fatty acid content) and the samples contained all essential amino acids. Total carotenoid content ranged from 3.2 to 48.0 μg g⁻¹, total flavonoid content (as catechin equivalents) from 1.9 to 4.5 mg g⁻¹, total reducing capacity (as gallic acid equivalents) from 7.0 to 15.0 mg g⁻¹, ferric-reducing antioxidant power from 2.8 to 8.9 μmol g⁻¹ and oxygen radical absorbance capacity (as Trolox equivalents) from 224.9 to 1117.0 μmol g⁻¹. CONCLUSIONS: This study reports for the first time a comprehensive chemical characterization of M. seminigra pot-pollen. The samples presented antioxidant activity, high values of nutrients and bioactive compounds such as polyphenols, carotenoids, insoluble dietary fibers, polyunsaturated fatty acids, and essential amino acids, comparable to other health foods.