BACKGROUND: Whey is one of the main agro-industrial by-products generated in the production of cheese. It is a residue with low pH and high content of organic and inorganic compounds, such as lactose, proteins, and minerals that can be used as nutrients in fermentation. The aim of the present study was to produce biomolecules with aroma and flavor properties by whey fermentation using Kluyveromyces marxianus (KM) and Debaryomyces hansenii (DH).RESULTS: These yeasts produced and accumulated in the culture broth aroma and flavor compounds, the main ones being ethanol, glycerol, propanoic acid, dihydroxyacetone, methionol, isopentanol, and 2-phenylethanol (2PE). This last one was retained as the target biomolecule, because of its potential to be commercialized industrially. Both yeasts were able to metabolize L-phenylalanine (Lphe) to produce 2PE, in monoculture and co-culture modes. When yeasts were used under monoculture mode, KM produced the highest 2PE concentration 82 ± 28 mg/L under aerobic fermentation, with yield of 0.16 ± 0.08 g 2PE /g Lphe and a productivity of 0.86 ± 0.18 mg 2PE /L*h at a fermentation time of 96 h. Whereas in co-culture mode the 2PE yield was 0.38 g 2PE /g Lphe , twice as high as the maximum yield for monocultures. This yield corresponded to a productivity of 1.93 ± 0.02 mg 2PE /L*h. CONCLUSION: The whey fermentation using KM and DH in co-culture mode is technically feasible. The KM yeast is apparently dominant and the co-culture of both yeasts led to increase in the 2PE yield and the productivity. The faster kinetics of KM quickly induced substrate starvation triggering early production and accumulation of 2PE.