Background/Objectives: Selenium biofortification is of interest for edible mushrooms, but excessive selenium (Se) may impair fungal growth and metabolic homeostasis. Methods: Agaricus bisporus mycelia were exposed to nominal sodium selenite (Na₂SeO₃) concentrations of 0–20 µg/mL, and phenotypic evaluation was integrated with transcriptomic and widely targeted metabolomic analyses of the 0, 5, 10, and 15 µg/mL groups. Results: At 5 µg/mL, final colony diameter and dry biomass were greater than those in CK (Tukey-adjusted p = 0.0313 and 0.0189, respectively), whereas 15–20 µg/mL reduced both endpoints. Transcriptomic results and exploratory metabolomic associations suggested concentration-related changes in starch and sucrose metabolism and glutathione-related redox processes. Phosphoglucomutase (PGM; gene1915) expression and associated sugar profiles were consistent with altered carbon metabolism; CUF1 (gene816) and RLM1 (gene5124) correlated with PGM as candidate regulators. Selenium exposure elicited isoform-specific glutathione S-transferase responses and induced expression of the glutathione peroxidase gene (GPX; gene6123). Conclusions: Low-dose sodium selenite (5 μg/mL) promoted Agaricus bisporus mycelial growth, whereas higher concentrations inhibited growth and were associated with changes in carbohydrate and glutathione-related metabolism.