This study investigated the physicochemical properties, antioxidant, and antitumor activities of melanin extracted from fungi associated with abundant marine macroalgae collected from Fayed, Suez Canal, Egypt during winter 2023. The epiphytic fungi included Aspergillus niger, Aspergillus flavus, Alternaria alternata, Cladosporium sp., and Curvularia chlamydospora. that are associated with the red algae Laurencia obtusa and Gracilaria verrucosa, as well as the green algae Cladophora crinalis, Enteromorpha compressa, and Ulva lactuca. Melanin exhibited a characteristic UV-Vis absorption peak at 235 mm. IR spectra of fungal melanin showed peaks at 3378 cm-1 and 3308 cm-1 (carboxyl, phenolic, hydroxyl, amine), 1720/1705 cm-1 (C=O), 1646/1610 cm-1 (C=C), and 1046/1040 cm-1 (C-O), indicating the presence of various functional groups. All fungal melanin pigments demonstrated significant antioxidant activity in a dose-dependent manner in both DPPH and ABTS assays. Aspergillus niger and Curvularia chlamydospore showed 50% activity at IC50 values of 12.2 and 13.48 µg/ml in the DPPH assay, and 15.47 and 25.87 µg/ml in the ABTS assay respectively. In vitro, cytotoxicity assays revealed that melanin exhibited differing levels of cytotoxicity against A549 lung cancer cells while showing negligible toxicity to normal cells at varying doses from 31.25 to 1000 µg/ml., significantly reducing tumor cell viability in a dose-dependent mode. Alternaria alternata demonstrated the highest cytotoxicity at low concentrations, signifying its potential as a promising anticancer agent. Our findings showed that fungi associated with macroalgae represented a novel source of melanin. This natural pigment exhibits significant potential for various applications, providing a strong theoretical foundation for its utilization.