Background: Aquaculture, particularly shrimp farming, has become an important economic sector in Viet Nam. The microalga Thalassiosira weissflogii is a valuable natural feed due to its high nutritional value and benefits for larval growth and water quality. Optimizing the culture medium is essential for enhancing biomass productivity and nutritional quality of this diatom for aquaculture applications. Objectives: This study aimed to identify the optimal nutrient medium for maximizing growth, biomass yield, and nutritional quality of the diatom Thalassiosira weissflogii. The comparative evaluation of different nutrient media was undertaken to provide a scientific basis for recommending a suitable medium for large-scale microalgal biomass production in aquaculture. Methods: The experiment involved culturing Thalassiosira weissflogii in four different nutrient media—AGP, Walne, F/2, and TT3—over an 8-day period, with three replicates per treatment. After cultivation, the algal biomass was analyzed for key nutritional components, including carbohydrates, carotenoids, and silica, to assess the effects of different nutrient media on algal nutritional quality. Results: The results revealed statistically significant differences among treatments (p < 0.05). The AGP medium exhibited superior performance, achieving the highest growth rate, maximum cell density (30 × 108 cells/mL on day 7), and the greatest mean biomass yield (3.21 ± 0.41 g/L). In terms of nutritional composition, AGP also produced the highest levels of carbohydrates (2.4 mg/0.1 g algae), carotenoids (0.68 µg/L), and silica (22.2%) compared with the other treatments. The Walne, F/2, and TT3 media resulted in lower growth performance and nutrient accumulation. Conclusion: The study demonstrates that the AGP medium is the optimal choice for culturing Thalassiosira weissflogii, meeting both high biomass productivity and superior nutritional quality requirements. This medium is therefore well suited for the production of natural live feed for seed production and commercial aquaculture.