<p>The Pacific white shrimp (<i>Penaeus vannamei</i>) industry faces significant challenges from diseases like Acute Hepatopancreatic Necrosis Disease (AHPND), driving the need for sustainable functional feed additives. This study investigated the effects of a mixed biomass of diatoms, <i>Thalassiosira weissflogii</i> and <i>Chaetoceros calcitrans</i>, as a dietary supplement for juvenile <i>P. vannamei</i>. Three isonitrogenous and isolipidic diets were formulated: a control diet with no microalgae, and two test diets containing the mixed biomass at 1% (Mix-Low) and 3% (Mix-High) inclusion levels. After a 60-day feeding trial, shrimp fed the microalgae diets showed significantly enhanced growth performance, with the Mix-High group achieving the highest specific growth rate (3.15 ± 0.12% day⁻¹), percent weight gain (132.5 ± 8.7%), and the lowest feed conversion ratio (1.42 ± 0.05). Furthermore, dietary supplementation significantly increased muscle protein content (21.7 ± 0.3% vs. 19.4 ± 0.1% in control). Upon challenge with <i>Vibrio parahaemolyticus</i> (AHPND strain), shrimp fed the control diet suffered 100% mortality within 144&#xa0;h, while shrimp fed both microalgae diets exhibited 100% survival, corroborated by histopathology showing no AHPND lesions. The results demonstrate that 1–3% inclusion of mixed diatom biomass acts as a potent functional feed additive, simultaneously enhancing growth, muscle quality, and disease resistance, offering a sustainable strategy for shrimp aquaculture.</p> Graphical Abstract <p></p>