Freshwater microalgae have attracted interest as an alternative food source of nutrients and bioactive compounds due to their sustainable production. This study aimed to evaluate the composition and bioaccessibility of amino acids (AA) and fatty acids (FA) present in the biomass of Chlorella vulgaris, Spirulina platensis, Scenedesmus acuminatus, Desmodesmus maximus, and Tetraselmis sp. Determination and quantification of AA and FA were carried out using High-performance liquid chromatography and Gas chromatography, respectively. Bioaccessibility was calculated by following the Minekus protocol and by comparing the concentration of each compound before and after gastrointestinal digestion. Statistical analysis (ANOVA, Tukey's and Student's t-test) considered p<0.05. The evaluated microalgae had a similar profile of AA and FA but varied (p<0.05) concentration. D. maximus had the highest total amount of AA (251.48 ± 0.30 mg/g). Lysine was the most abundant essential amino acid (EAA) detected in all microalgae (18.74 – 33.15 mg/g). S. platensis showed the lowest bioaccessibility for threonine (4.85%), while D. maximus and Tetraselmis sp. showed the highest bioaccessibility for tryptophan (~257%). Seven FA were detected in the microalgae studied, including 3 saturated fatty acids (myristic, palmitic and estearic acids), 2 mono-unsaturated fatty acids (palmitoleic and oleic acids) and 2 polyunsaturated fatty acids (linoleic and linolenic acids). Palmitic and oleic (ω9) acids were the FA detected in higher concentrations (39.00 – 62.23 µc/g and 7.32 – 27.15 µc/g, respectively). Linoleic (ω6; 5.37 – 11.35 µc/g) and linolenic (ω3; 4.54 – 7.04 µc/g) acids were also detected. The bioaccessibility was higher than 70% for most FA compounds in all studied species. The results obtained showed that the studied microalgae can provide bioaccessible EAA and essential FA (ω3, ω6 and ω9) needed by humans. Findings encourage their application in the development of new functional foods carrying EAA and important FA, expanding the commercial potential of microalgae-based products.