Sooro Renner Nutrição S.A. company is found in the Western Region of Paraná/Brazil, which is highlighted nationally and in Latin America concerning the production of whey protein concentrate (WPC). During the production of WPC, performed in ultrafiltration membranes, the subproduct cheese whey permeate (CWP) is generated, which is rich in nutrients, such as lactose, minerals, and vitamins. This subproduct is reported as a potential culture medium to grow microorganisms. Thus, this research, performed in partnership with the Sooro company, aimed to develop biotechnological products employing sequential fermentations to fully use this subproduct bioconverting the ethanol obtained from CWP into vinegar employing the acetic bacterium Acetobacter aceti and different methods - Orleans, aerated, and stirred. The biotransformation into ethanol was performed by Kluyveromyces marxianus (alcoholic fermentation step) using a 2³ factorial experimental design to investigate the influence of lactose concentration, temperature, and pH. The maximum ethanol production was 47.18±0.05 g L-1, employing the conditions 88 g L-1 of lactose, 29 °C, and pH 4.5 in 45 h. Besides ethanol, probiotic cellular biomass, prebiotic galacto-oligosaccharides, and organic acids were also produced. In the oxidation stage, the Orleans method presented the best production: 42.30±0.08 g L-1 of acetic acid in 21 days. After this production, reductions of chemical oxygen demand and biochemical oxygen demand of the CWP were 60 and 65%, respectively. The results showed the great potential of CWP as a fermentation medium to obtain biotechnological products as a rentable and viable alternative to fully use CWP.