In the context of increasing demand for food products, microalgae garnered considerable interest due to their essential roles in supporting aquatic food webs and serving as bait in aquaculture. In this regard, it is crucial to clarify their optimal culturing conditions, including both single-factor and multiple-factor combinations. In this study, Thalassiosira weissflogii and Pyramimonas sp. were cultivated under five levels of temperature, light intensity, and nitrate to determine their optimum growth conditions. A full factorial experiment was then conducted, employing two levels for each factor (10 and 25 degrees C for temperature; 50 and 150 mu mol photons m-2 s-1 for light intensity; 2 and 100 mu mol L-1 for nitrate concentration) to investigate the growth, photosynthesis, and amino acid and fatty acid compositions of the two species. T. weissflogii exhibited an optimal growth temperature 4 degrees C higher than that of Pyramimonas sp., while the latter performed better at low temperatures. The full factorial experiment revealed that temperature was the most significant factor influencing the growth of two species. The fatty acid composition of T. weissflogii was primarily affected by temperature changes, with an average 4 % higher percentage of polyunsaturated fatty acids observed at high temperature. Nitrate concentration emerged as the key factor influencing photosynthetic rates, as well as soluble protein and carbohydrate contents, while it was the only factor affecting amino acid profiles. Generally, nitrate limitation resulted in lower photosynthetic rates, higher soluble carbonate contents, while soluble protein content and amino acid composition varied depending on the species and culturing conditions. These findings provide valuable information and reference points for enhancing biomass accumulation and the production of active substances in microalgae cultivation.