Commercial wine fermentation is commonly conducted by inoculated strains of Saccharomyces cerevisiae. However, other non-Saccharomyces yeast species have recently become popular co-inoculants. Co-inoculated yeast species compete with each other for nutrients, and such competition may impact fermentation kinetics and aroma production. Understanding the specific nutrient requirements of non-Saccharomyces strains therefore is essential to better characterize the competitive potential of each strain, and to support rational decision making for nutrient supplementation during wine making. This study investigated the nitrogen source preference of commercial non-Saccharomyces yeasts by conducting pure culture and sequential culture fermentations in synthetic grape musts with adjusted nitrogen contents. Amino acid and ammonium uptake varied between yeast species. Lachancea thermotolerans and Torulaspora delbrueckii assimilated more nitrogen at a faster rate than Pichia kluyveri and Metschnikowia pulcherrima. Significant variation in amino acid preference between species was observed. Sequential fermentations confirmed the more competitive behaviour of L. thermotolerans and T. delbrueckii, with consequences for fermentation kinetics and aroma production. Furthermore, the data suggest that declining populations of non-Saccharomyces yeasts release nitrogen and supports the activity of S. cerevisiae. The data provide the most detailed assessment of nitrogen utilisation by the investigated yeast strains in a wine environment.