The yeast Yarrowia lipolytica is well known for its versatile production of metabolites from various substrates, but, although isolated from, e.g., wild-fermented Belgian Sour beers, it is rarely considered a starter culture in fermented beverages. In this study, we aimed to elucidate the ability of Y. lipolytica to ferment brewers’ wort containing iso-α-acid for 7 days at low and high aeration and at 20 °C and 30 °C, with a special focus on amino acid consumption and production of volatile aroma compounds. Y. lipolytica was able to grow in the wort under all four conditions, although the growth was inhibited. Furthermore, it only consumed glucose and fructose, and no ethanol was formed. Moreover, under high aeration conditions, Y. lipolytica consumed 75–80% of the amino acids in the wort. Interestingly, no esters were produced during the fermentations, and only five higher alcohols (1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-methyl-3-buten-1-ol, and 2-phenylethanol), two aldehydes (3-methylbutanal and (E)-2-nonenal), two ketones (cyclopentanone and 9-oxabicyclo [6.1.0]nonan-4-one), one fatty acid (3-methyl-butanoic acid), and one benzene derivate (1,2,4-trimethyl-benzene) were produced. These results may contribute to the potential use of Y. lipolytica in a traditional brewery for the production of novel beers; e.g., alcohol-free beer.