Third-generation biomass production utilizing microalgae exhibits sustainable and environmentally friendly attributes, along with significant potential as a source of physiologically active compounds. However, the process of screening and localizing strains that are capable of producing high-value-added substances necessitates a significant amount of effort. In the present study, we have successfully isolated the indigenous marine diatom Odontella aurita OAOSH22 from the east coast of Korea. Afterwards, comprehensive analysis was conducted on its morphological, molecular, and biochemical characteristics. In addition, a series of experiments was conducted to analyze the effects of various environmental factors that should be considered during cultivation, such as water temperature, salinity, irradiance, and nutrients (particularly nitrate, silicate, phosphate, and iron). The morphological characteristics of the isolate were observed using optical and electron microscopes, and it exhibited features typical of O. aurita. Additionally, the molecular phylogenetic inference derived from the sequence of the small-subunit 18S rDNA confirmed the classification of the microalgal strain as O. aurita. This isolate has been confirmed to contain 7.1 mg g−1 dry cell weight (DCW) of fucoxanthin, a powerful antioxidant substance. In addition, this isolate contains 11.1 mg g−1 DCW of eicosapentaenoic acid (EPA), which is one of the nutritionally essential polyunsaturated fatty acids. Therefore, this indigenous isolate exhibits significant potential as a valuable source of bioactive substances for various bio-industrial applications.