Banana peels account for about 61 million tons of waste each year globally. The aim of this project was to investigate the possibility of using banana peels as a substrate to cultivate edible filamentous fungi. The peels were subjected to physical and thermal pretreatments while variables such as changes in the medium pH, biomass concentration, fungal strain dependence, and protein content of the fungal biomass were analyzed. The experiments were carried out in three phases. The purpose of phase I was to identify which of the four fungal strains among Neurospora intermedia, Aspergillus oryzae, Rhizopus oryzae, and Rhizopus oligosporus could grow in a medium containing ball-milled banana peel powder (BPP) only. In phase II, the best performing strains from phase I in terms of biomass concentration, i.e., A. oryzae and R. oryzae, were cultivated using banana peel broth (BPB) obtained from thermal pretreatment of BPP. During this phase, the impact of medium supplementation with yeast extract was also assessed. The biomass yield for A. oryzae and R. oryzae 2.9 g/L and 1.6 g/L, respectively, yeast supplementation compared to 2.7 g/L and 0.7 g/L, respectively, without supplementation. In phase III, the experiments performed in phase II without yeast extract supplementation were scaled up, after which protein analysis was performed. A crude protein content of 8.82% was determined for A. oryzae, while in R. oryzae, a higher value of 21.1% was obtained. The protein content from both fungal strains was much higher than that present in the BPP, which was 4.8 g/L. The results showed the potential of using banana peel as a substrate to produce edible fungal biomass with higher protein content and thus has potential applications as animal feed or human food. Further studies are needed to optimize the process in order to raise the fungal biomass yield as well as increase the protein content of the biomass. In addition, comprehensive characterization of the fungal biomass would reveal other important components, such as the amino acid profile.