The global food industry is facing significant challenges due to population growth, climate change and shifting consumer preferences. These factors contribute to the increasing demand for sustainable food production and food waste management. The valorisation of food by-products through microbial conversion represents a viable solution to these issues. This study explores the use of filamentous fungi, specifically Aspergillus oryzae and Fusarium venenatum, to convert food waste into valuable products such as mycoprotein. These fungi present a Generally Recognized as Safe (GRAS) status with diverse enzymatic capabilities to grow on diverse substrates. This research focuses on evaluating their potential to produce protein-rich biomass from different local by-products such as carob, rice, and tiger nut, obtained from the production of vegetable beverages, under different preparation methods, including sterilization, crushing or not, hydration with sterile water, and supplementation with 4% glucose and 2% yeast extract. The results show that in carob and tiger nut by-products, the fungi were able to grow on the uncrushed substrates; however, in the rice by-products, the fungi needed the crushing and supplementation of the substrates to grow efficiently. The protein content of the fungal biomass was analyzed using the Kjeldahl method. The results indicate significant differences in protein content based on the substrate and preparation method. Specifically, the supplementation of crushed substrates led to the highest protein yields in both fungi when cultured on rice. These results highlight the efficacy of using food waste substrates for fungal protein production, which aligns with Sustainable Development Goals and the circular economy. Furthermore, this obtained fungal biomass could be used as a basis for the development of meat analogues. The findings have implications for reducing environmental impact, enhancing food security, and promoting innovative solutions for waste management in the food industry.