<p>The yeast-mediated enrichment represents a biotechnological approach for improving the nutritional and functional properties of microalgal biomass. This study aimed to evaluate and optimize incubation parameters to improve the protein content and antioxidant capacity of <i>Porphyridium cruentum</i> biomass while supporting the growth of the yeast <i>Kluyveromyces marxianus</i>. The individual and interactive effects of incubation time (24, 48, and 72&#xa0;h), inoculum concentration (2%, 6%, and 10% v/v), and glucose supplementation (0%, 1%, and 2% w/v) were assessed on protein content, antioxidant capacity (ORAC and DPPH), and yeast cell density. Subsequently, Response Surface Methodology (RSM) using a Box-Behnken Design (BBD) was applied to maximize these responses. Results demonstrated significant improvements in protein content and antioxidant capacity under specific treatments. The protein content reached 50.69 ± 0.29% (a 17.9% increase compared to raw biomass). Antioxidant capacity also improved, highlighted by an 89% increase in ORAC values compared to raw biomass. Robust yeast proliferation strongly correlated with the increased protein, particularly under glucose-supplemented conditions, indicating that exogenous carbon promoted yeast biomass accumulation, contributing to the observed protein enrichment. Optimal conditions were: 44&#xa0;h incubation, 6.2% inoculum, and 1.2% glucose, yielding validated protein content of 49.76 ± 2.69%, antioxidant capacity values of 11.57 ± 0.64 × 10³ (ORAC), 3.11 ± 0.33 × 10³ (DPPH) µM Trolox equivalents per 100&#xa0;g, and 8.33 ± 0.07 Log CFU mL⁻¹ yeast density. Although the underlying mechanisms were not directly evaluated, this study establishes glucose-supplemented yeast-mediated enrichment as a viable strategy for producing protein-enriched and antioxidant-rich biomass, with potential applications in food and feed systems.</p>