Halamphora sp., a benthic diatom has been known to improve growth and nutritional value of aquatic animals. Biofloc, a zero-water exchange system consisting of multiple organism aggregates could act as natural food in an aquaculture system. It could improve aquaculture productivity through enhanced nutritional values leading to increased growth and survival. Biofloc also reduces pathogen proliferation, improving animal immunity and enhancing biosecurity. Addition of probiotics such as microalgae and bacteria in biofloc system can further enhance the nutritional profile and health aspects of shrimp culture. This study aims to assess the impact of adding a microalga Halamphora sp. on the growth, water quality, disease resistance, and nutritional composition of the Black tiger shrimp (Penaeus monodon) grown in rice bran biofloc system. The evaluation of P. monodon’s growth performance such as the final weight, specific growth rates (SGR), feed conversion ratios (FCR), and survival rates were performed weekly. The nutritional composition of both P. monodon and the biofloc, including their FAME components, were also determined. Water quality parameters were concurrently monitored each week. Sub-samples of P. monodon were also challenged with Vibrio parahaemolyticus and mortality rates for each treatment were recorded. The results showed that P. monodon treated with rice bran biofloc and Halamphora sp. showed higher growth and survival rates compared to controls without any microalga and carbon source addition. The Halamphora sp. addition in rice bran biofloc in P. monodon culture also improves water quality parameters, including nitrate, nitrite, and orthophosphate level. Enhanced total suspended solids (TSS) and floc volume (FV) was also observed with improved nutrient content such as the essential fatty acids that include PUFA, Omega-3, MUFA, Omega-6, and DHA. Challenge test result showed lower shrimp mortality rates in treatment added with the microalgae and rice bran. In the histological examination of challenged P. monodon’s hepatopancreas from the control group, more intense hepatopancreatic cell lesions and necrotic cells were noted while P. monodon treated with rice bran biofloc and Halamphora sp. were observations of sloughed epithelial cells in the tubule lumens and haemocyte infiltration, with minimal lesions. In summary, Halamphora sp. addition to a biofloc system with rice bran improves P. monodon post larvae and could potentially lead to the development of more sustainable and cost-effective practices in shrimp farming. Furthermore, the reduction in pathogen proliferation could minimize the reliance on antibiotics, leading to a healthier and safer product for consumers.