Food industry by-products such as grape pomace (GP), tomato pomace (TP), and spent coffee grounds (SCG) are rich in polyphenols (PP) but are easily biodegradable. The aim of this study is to test Spontaneous Fermentation (SF) as treatment to modify PP profile and bioactivity. The results highlighted that the by-products' organic matter and the microbial populations drove the SF evolution; heterolactic, alcoholic, and their mixtures were the predominant metabolisms of TP, GP, and SCG + GP co-fermentation. Increases in the extractable amounts and antiradical activity occurred for all the biomasses. Regarding the aglycate-PPs (APP), i.e. the most bioreactive PPs, significant changes occurred for TP and GP but did not influence the anti-inflammatory bioactivity. The co-fermentation increased significantly chlorogenic acid and consumed most of the APPs, acting as a purification system to obtain a highly concentrated APP fraction, so that the extract might be employed for a specific purpose.