The aim of this study was to investigate the behaviour of a Serratia marcescens-Kluyveromyces fragilis mixed culture in whey, with the objective of proteases production and organic waste reduction.Discontinuous aerobic fermentations in whey were carried out using individual pure cultures and mixed cultures of S. marcescens and K. fragilis. Cell growth, protease production, lactose and proteins consumption and COD/TOC reduction were monitored. Lactose and protein content of the fermenting medium was almost depleted in the mixed cultures, achieving a reduction in the organic content much higher than in both pure cultures. Interestingly, proteolytic activity in the mixed cultures was similar to that obtained for S. marcescens in pure culture. In addition, protease stability was increased in the mixed cultures. Kinetic models were developed fitting well with the experimental results.Mixed cultures were found to maintain the achievements of each individual fermentation, yielding a high and stable production of proteases and a significant reduction of COD/TOC.Mixed cultures tested in this work have shown a synergistic effect with possible industrial applications. These results lead to a gain in the chain value for enzyme production with an environmentally friendly operation.