The rapid growth of aquaculture and the lack of fish meal demand new sustainable ingre-dients. Although fungal biomass is found to be a promising sustainable fish feed supplementation candidate, the characteristics of this protein-rich source are closely influenced by the quality of the applied growth medium. In this work, the nutritional properties of pure filamentous fungal biomass provided from the cultivation of Aspergillus oryzae, Neurospora intermedia and Rhzopus oryzae were evaluated to assess their potential as alternative novel protein sources in fish feed. In this regard, fungal biomass yields of up to 0.19 ± 0.005 (g dry biomass/g substrate glucose) were obtained during submerged cultivation of fungal strains. The pure fungal biomass acquired could contain significant amounts of protein up to 62.2 ± 1.2% (w/w). The obtained protein had a high quality with notable inclusion of essential amino acids such as lysine, arginine, methionine and threonine with comparable concentrations to those of fish meal. Fungal biomass is mainly considered as protein source, however, entitlement of 6.9 ± 0.5, 4.0 ± 0.7 and 17.2 ± 1.1% (w/w) of lipids and ratio of polyunsatu-rated fatty acids (PUFA) to saturated fatty acids (SFA) of 1.37:1, 1.74:1 and 1.47:1 in A. oryzae, N. intermedia and R. oryzae, respectively, signal health benefits for the fish. Considering the results, protein-rich pure fungal biomass with amino acid composition is greatly compatible with fish meal, and contains essential nutrients such as fatty acids and minerals. This pure biomass constitutes a promising sustainable alternative supplement to be introduced in fish feed industry.