The study presented here aims at identifying the source of redox mediators (riboflavin), electron carriers nicotinamide adenine dinucleotide (NAD) and carbon to perform decolorization of azo dye under anaerobic conditions after osmotic shock pretreatment of residual yeast from industrial fermentation. Pretreatment conditions were optimized by Doehlert experiment, varying NaCl concentration, temperature, yeast density and time. After the optimization, the riboflavin concentration in the residual yeast lysate (RYL) was 46% higher than the one present in commercial yeast extract. Moreover, similar NAD concentration was observed in both extracts. Subsequently, two decolorization experiments were performed, that is, a batch experiment (48 h) and a kinetic experiment (102 h). The results of the batch experiment showed that the use of the RYL produced by the optimized method increased decolorization rates and led to color removal efficiencies similar to those found when using the commercial extract (∼80%) and from 23% to 50% higher when compared to the control (without redox mediators). Kinetics analysis showed that methane production was also higher in the presence of yeast extract and RYL, and biogas was mostly generated after stabilization of color removal. In all kinetics experiments the azo dye degradation followed the pseudo-second-order model, which suggested that there was a concomitant adsorption/degradation of the dye on the biomass cell surface. Therefore, results showed the possibility of applying the pretreated residual yeast to improve color removal under anaerobic conditions, which is a sustainable process.