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BACKGROUND Vegetable oil is more difficult for microorganisms to degrade compared with carbohydrates and protein. Oil‐rich waste creates serious environmental and health concerns if it remains untreated. The aim of the work reported here was to evaluate the effects on the nutritional composition of filamentous fungal biomass by growing it in pure olive oil as a carbon source. RESULTS The growth of different filamentous fungal strains (Aspergillus oryzae, Neurospora intermedia and Rhizopus oryzae) was investigated in pure olive oil. A pre‐germination stage of either glucose or yeast extract was necessary for successful fungal growth in olive oil. A. oryzae showed superior performance in comparison with N. intermedia and R. oryzae in terms of biomass growth. The obtained biomass from A. oryzae and N. intermedia was analyzed for protein, fat, ash and alkali‐insoluble material, where the presence of olive oil had a steering effect on biomass growth (16 g L−1 with oil versus 4 g L−1 without oil). Nutritional composition of the fungal biomass of A. oryzae contained 0.33% fat and 48% protein when cultivated in medium without olive oil supplementation, while 31% fat and 14% protein contents were observed in the presence of olive oil‐containing medium. Similar trends for fat and protein contents were observed for the biomass of N. intermedia. Moreover, microscopy confirmed the presence of oil globules inside the fungal cells. CONCLUSIONS Fat composition of fungal biomass can be steered through addition of olive oil, which increases the versatility of the produced biomass for various applications, namely in feed, food and biofuel production. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).