<![CDATA[Graphical Abstract Highlight Research 1. Three indigenous Indonesian Euglena gracilis strains were identified based on a morphological and molecular approach through the ITS2 region. 2. E. gracilis IDN 22 exhibited the highest specific growth rate. 3. E. gracilis IDN 22 produced the highest amounts of biomass, protein, paramylon, and lipids under the tested conditions, making it the most promising strain. 4. Euglena gracilis IDN 22 exhibited the highest DHA content among the three strains. DHA is an omega-3 fatty acid that has significant potential as a functional food ingredient. Abstract Acidophilic microalgae represent a promising source of novel food ingredients due to their unique biochemical properties. This study aims to identify and screen the indigenous tropical Euglena gracilis strains from three Indonesian aquatic habitats, evaluating their growth, nutritional profiles, and fatty acid composition to assess their potential. Three E. gracilis strains (IDN 1, IDN 22, and IDN 40) were isolated from Indonesian aquatic habitats and characterized using morphological and ITS2 molecular analyses. Growth performance was determined by cell counting, biomass production by gravimetric analysis, protein content using the Bradford assay, lipid content following the Bligh and Dyer method, and fatty acid composition by gas chromatography with flame ionization detection (GC-FID). This study is the first to integrate morphological, molecular (ITS2), and biochemical characterization to evaluate native Indonesian E. gracilis strains comprehensively. Among these strains, IDN 22 was the most superior. This strain exhibited a growth rate of 0.202 ± 0.006, a biomass yield of 0.558 ± 0.072 g/L, a protein content of 520.171 ± 12.324 µg/mL, a lipid accumulation of 15.33 ± 0.015%, and a paramylon concentration of 51.0 ± 1.80%. While total fatty acid content ranked as IDN 1 < IDN 22 < IDN 40, IDN 22 notably produced the highest levels of docosahexaenoic acid (DHA) among the three strains. These findings establish a scientific basis for selecting superior indigenous E. gracilis strains and demonstrate the potential of integrated strain characterization to support the development of functional foods, nutraceuticals, and other sustainable microalgal bioproducts. ]]>
