This work aimed at using a pool of selected enterococci and fungal proteases to hydrolyse wheat gluten during long-time fermentation. A liquid dough made with wheat flour (20% w/w) was fermented with three Enterococcus strains (dough A) or with the combination of enterococci and Rhizopus oryzae proteases (dough B). After 48 h of fermentation, dough A and B had a concentration of water-soluble peptides approximately threefold higher than the chemically acidified dough (CAD), used as the control. The same was found for the concentration of free amino acids, being higher in dough B with respect to dough A. SDS-PAGE analysis showed that albumin and glutenin fractions were partially hydrolysed, while gliadins almost disappeared in dough A and B, as confirmed by two-dimensional electrophoresis, RP-HPLC and R5-ELISA analyses. The combined use of enterococci and fungal proteases showed a decrease of the gluten concentration of more than 98% during long-time fermentation. The use of the mixture of selected enterococci and R. oryzae proteases should be considered as a potential tool to decrease gluten concentration in foods.