Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.