The increasing demand for safer and environmentally sustainable products has intensified the search for natural alternatives to synthetic dyes. Filamentous fungi are promising sources of natural pigments due to their metabolic diversity and the feasibility of large-scale production. In this study, filamentous fungi isolated from Amazonian freshwater environments were evaluated for their potential to produce natural pigment-associated metabolites under different nutritional conditions. Forty-five fungal isolates were screened in solid media and subsequently cultivated in submerged fermentation using three media: potato dextrose broth supplemented with yeast extract (BD + YE); malt extract broth (ME); and yeast extract–sucrose broth supplemented with magnesium sulfate (YES). Among the 39 pigment-producing isolates, seven were selected for further investigation. Sucrose favored the highest absorbance values of pigment extracts, particularly for isolates identified as Talaromyces amestolkiae. In addition, the extract of T. amestolkiae TA10P5-3 exhibited the highest absorbance value (6.83 abs. units at 400 nm) when cultivated in YES medium, indicating stronger chromophore-associated spectral signals. This extract also showed antimicrobial activity against Pseudomonas aeruginosa (625 μg/mL), Staphylococcus epidermidis (312 μg/mL), and Candida tropicalis (625 μg/mL). Finally, the TA10P5-3 extract presented high total phenolic content (246.30 mg GAE/g) and antioxidant activity (EC₅₀ = 5470 μg/mL). These findings highlight Amazonian freshwater fungi as promising sources of natural pigments with potential industrial applications.