Biquinho pepper (Capsicum chinense) is a native species of Brazilian flora that is widely appreciated as a culinary condiment. Additionally, people have taken up the increasing concept of using natural food to manage health. Thus, this study aimed to characterize biquinho pepper flour by its chemical composition, mainly considering its profile of fatty acids and phytosterols. The peppers were dried in an air circulation oven at 40 ° C for 48 h and ground in a domestic processor. Then, the material was passed through a 24-mesh sieve to obtain the pepper flour. Regarding the centesimal composition, the following results were found: moisture (9.49 g/100 g), lipids (5.09 g/100 g), proteins (8.37 g/100 g), carbohydrates (51.22 g/100 g), fibers (20.21 g/100 g), and ash (5.60 g/100 g). Minerals were also determined, showing magnesium as the most abundant (239.60 ± 0.04 mg/100 g), followed by calcium (176.05 ± 0.05 mg/100 g). To characterize the fatty acids profile, the lipid fraction of samples was converted into methyl esters by transesterification and analyzed using a gas chromatograph (Shimadzu GC 2010, Tokyo, Japan). The main fatty acids determined were: linoleic (C18: 2 n6c, 57.66 ± 0.66 g/100 g), palmitic (C16:0, 12.95 ± 0.29 g/100 g), and oleic acids (C18:1 n9c, 5.71 ± 0.04 g/100). The sum fatty acid levels decreased in the order of polyunsaturated (59.79 g/100 g), saturated (17.66 g/100 g), and monounsaturated (9.58 g/100 g). Phytosterols were extracted by direct saponification. Samples and standards were derivatized to thrymethylsilyl TMS ethers and evaluated by gas chromatograph analysis. Two phytosterols were determined: β-sitosterol (40.29 ± 0.83 mg/100 g) and stigmasterol (21.18 ± 0.64 mg / 100 g). Thus, this study highlights C. chinense as a source of functional phytochemicals. Indeed, in addition to its commercial and economic value, biquinho pepper has significant health-promoting potential.