Vegetable oil is more difficult to degrade by microorganisms in comparison to carbohydrates and protein. Thus, it creates serious environmental and health concerns if oil-derived waste streams produced by restaurants and industries remain untreated. In this study, a strategy has been developed to grow filamentous fungi in pure olive oil so that it can be used as a bench-mark for growth in olive oil mill sidestreams. The growth of different strains (Aspergillus oryzae, Neurospora intermedia and Rhizopus oryzae) was tested in pure olive oil. A pre-germination stage using glucose as carbon and energy source, or the addition of yeast extract, were found necessary for successful fungal growth in olive oil. Here, A. oryzae showed a superior performance in comparison to N. intermedia and R. oryzae. Medium pH did not impact A. oryzae growth in olive oil, whereas a concentration higher than 40 g/L of the latter impaired the growth of the ascomycete. Obtained biomasses from A. oryzae and N. intermedia cultivations in olive oil were analyzed and compared for protein, fat, ash, and alkali-insoluble material (cell wall content), where the presence of olive oil had a steering effect. The fungal biomass of A. oryzae, obtained from cultivation in the absence of olive oil, contained 0.33% fat and 48% protein, whereas the respective values in the presence of olive oil were 31% and 14%. Similar trends on fat and protein contents were observed for the biomass of N. intermedia. Sudan black staining was also performed on fresh biomass which clearly indicated the presence of oil globules inside the fungal cells. This research can be a fundamental step towards treatment of oil-based waste streams, which entails high-energy and costs if treated, or environmental impacts during informal discharges. Moreover, the fact that the composition of fungal biomass can be steered through addition of olive oil increases the versatility of the originated biomass for various applications, namely in feed, food and biofuel production.