Endophytic fungi are recognized for their capacity to produce bioactive metabolites, yet their metabolic output is strongly modulated by preservation strategies. This study evaluated the influence of three long-term preservation methods—PDA slants with mineral oil (PB), Castellani’s method (PC), and filter paper with mineral oil (PF)—on the production of phenolic compounds and antioxidant activity by the Amazonian endophytes Cophinforma mamane and Aspergillus niger. Post-reactivation morphological analyses revealed method-dependent changes in colony pigmentation, texture, and sporulation intensity. PB preservation induced the most vigorous growth and enhanced sporulation in A. niger. For extracellular metabolites (AcOEt extracts), PB yielded the highest total phenolic content in A. niger (258.24 mg GAE/g), whereas PF and PC favored C. mamane (154.48 and 153.33 mg GAE/g). For intracellular ethanolic extracts, PB was most effective for C. mamane, producing 206.19 mg GAE/g for total phenols and the highest antioxidant activities (EC₅₀ = 1.40 mg/mL and FRAP = 95.61 µmol TE/g). HPLC–DAD identified protocatechuic acid as the predominant compound, which was especially abundant in PC and PF-preserved A. niger (60.76 and 85.38 µg/mg, respectively). Multivariate correlations indicated syringic (r = 0.53) and p-coumaric (r = 0.63) acids as the main contributors to antioxidant potential. These findings demonstrate that preservation methods may positively or negatively modulate fungal biosynthetic capacity, underscoring the importance of species-specific preservation protocols for maintaining metabolic stability in culture collections.