Microalgae are considered a sustainable source of high-value products with health benefits. Marine algae-derived omega-3 long-chain polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are considered dietary elements with effects on mental health, cognition enhancement, and cardiovascular protection. This study investigated the temperature effect on omega-3 LC-PUFA production in eight species of microalgae from various taxonomic groups, with a focus on achieving an optimal balance between omega-3 accumulation and efficient growth performance. Samples were batch-cultivated at four different temperatures, with constant light, and fatty acid methyl esters (FAME) were analyzed by gas chromatography. Several nutritional indices were calculated to assess the potential value of biomass produced for human consumption. Two promising candidates were identified suitable for batch cultivation and large-scale production: Nannochloropsis oculata for EPA and Isochrysis galbana for DHA production, with optimum productivities obtained between 14 and 20 °C, and nutritional indices falling within the range required for nutritional benefit.