<p>In order to evaluate the directly waterborne inoculation of marine red yeast (<i>Rhodotorula mucilaginosa</i>) on the aquaculture of <i>Penaeus vannamei</i> with zero-water exchange biofloc technology (BFT), this study assessed the effects of dosing <i>R. mucilaginosa</i> at four levels (T0, 0; T1, 2 × 10³ cells/mL; T2, 2 × 10⁴ cells/mL; T3, 2 × 10⁵ cells/mL). Yeast was supplemented every 4&#xa0;days. A total of 91 shrimp (initial body weight, 6.01 ± 1.31&#xa0;g; body length, 9.79 ± 0.85&#xa0;cm) were stocked per 350-L tank and reared for 35&#xa0;days. The experiment consisted of 12 aquaculture tanks (three replicates per group). The farming benefits were assessed by analyzing variations in the microbial community structure and nutritional composition. The highest dose (T3) produced a higher final body weight than all other treatments (<i>P</i> &lt; 0.05), whereas differences in the remaining growth metrics were not statistically significant (<i>P</i> &gt; 0.05). Supplementation with a higher dose (T3) of <i>R. mucilaginosa</i> allowed its colonization within the biofloc ecosystem, reducing eukaryotic microbial diversity without significantly affecting the prokaryotic diversity. Nutrient analysis showed increased whole-body astaxanthin and higher crude protein as well as essential and flavor amino acids in both bioflocs and shrimp muscle, with the strongest responses at T3 (<i>P</i> &lt; 0.05); crude lipid did not change (<i>P</i> &gt; 0.05). Hepatopancreatic trypsin, lipase, and amylase activities rose with dose, with T3 exceeding the other groups (<i>P</i> &lt; 0.05). Furthermore, the activities of digestive enzymes (trypsin, lipase, amylase) exhibited a dose-dependent increase, with the T3 group showing significantly higher enzymatic activities compared to other groups (<i>P</i> &lt; 0.05). In conclusion, the waterborne inoculation of <i>R. mucilaginosa</i> modulated the biofloc microbial composition, nutrition, and growth of juvenile <i>Penaeus vannamei</i>.</p>