<p>Microalgae have diverse applications in food, animal feed, biofuels, fertilizers, cosmetics, nutraceuticals, and pharmaceuticals, but challenges persist in improving nutrient utilization in large-scale farming. Nitrogen and temperature significantly influence growth rates and nutritional composition in microalgae. The study examined the effects of temperature (15, 20, 25, 30, and 35 °C) and nitrogen levels (36, 72 and 288 mg L⁻¹) on growth, protein content, lipid accumulation, and fatty acid composition of two freshwater microalgae, <i>Chlorella sorokiniana</i> and <i>Scenedesmus quadricauda</i>, during the exponential and stationary phases, cultivated in modified Conway medium. The specific growth rate of <i>C. sorokiniana</i> peaked at 1.31 day⁻¹ under a nitrogen concentration of 72 mg L⁻¹, with temperature having a more pronounced effect than nitrogen. For <i>S. quadricauda</i>, the growth rate reached 1.43 day⁻¹ at 72 mg L⁻¹ but declined significantly at higher nitrogen concentrations and temperatures above 25 °C. Biochemical analyses revealed that optimal cellular carbon, nitrogen, and phosphorus contents were achieved at 72 mg L⁻¹ nitrogen and 25 °C in both species. Protein content also peaked under these conditions during the exponential phase, reaching 54.75% in <i>C. sorokiniana</i> and 65.30% in <i>S. quadricauda</i>. Lipid content was highest at 288 mg L⁻¹ nitrogen and 25 °C. Fatty acid profiles highlighted the dominance of palmitic acid, oleic acid, and α-linolenic acid (ALA). Both species showed the highest ALA content at 72 mg L⁻¹ nitrogen and 25 °C, with 21.90% in <i>C. sorokiniana</i> and 25.30% in <i>S. quadricauda</i> during the stationary phase. The findings emphasize the critical role of nitrogen levels and temperatures in optimizing growth and biochemical yields for microalgae-based applications.</p>