The bakery industry generates a substantial food waste, leading to global environment pollution and economic losses. However, these by-products contain valuable bioactive compounds, such as melanoidins, which are brown pigmented compounds with significant antioxidant, antimicrobial and anti-inflammatory properties, making them ideal functional ingredient. This study aimed to evaluate the optimal enzymatic extraction of melanoidins from bread crust using different proteolytic enzymes: pronase E, serine-endo-protease (SP), metalloenzyme protease (MP) and endoprotease papain enzyme (EP). While the yield of melanoidins from SP and EP was lower than that of Pronase E, it was significant. No cytotoxicity of bioaccessible fractions, gastrointestinal and colonic fraction, were observed in the cell lines Caco-2, HUVEC and SHSY5Y. The antioxidant properties, chelating capacity and ability to stabilize hydroxyl radical were characterized in bioaccesible fractions of the four melanoidins extracts. Moreover, melanoidins showed positively influenced on the intestinal microbiota by increasing beneficial bacteria like Bifidobacterium and Lactobacillus, while reducing non-beneficial bacteria such as Bacteroides spp. and Clostridium leptum, with particularly significant results for SP and EP enzymes extracts. In conclusion, SP and EP enzymes appear to be viable alternatives to Pronase E for obtaining melanoidin extracts as functional ingredients. These enzymes offer potential cost advantages and contribute to the sustainable utilization of bakery by-product melanoidins in various food applications. Implementing these alternatives could help reduce food waste, environmental pollution, and give add value to the bakery industry while promoting the use of valuable bioactive compounds.