Alcohol-free beer (AFB) is no longer just a niche product in the beer market. For brewers, this product category offers economic benefits in the form of a growing market and often a lower tax burden and enables brewers to extend their product portfolio and promote responsible drinking. Non-Saccharomyces yeasts are known for their flavor-enhancing properties in food fermentations, and their prevailing inability to ferment maltose and maltotriose sets a natural fermentation limit and can introduce a promising approach in the production of AFB (≤0.5% v/v). Five strains isolated from kombucha, Hanseniaspora valbyensis, Hanseniaspora vineae, Torulaspora delbrueckii, Zygosaccharomyces bailii and Zygosaccharomyces kombuchaensis were compared to a commercially applied AFB strain Saccharomycodes ludwigii and a Saccharomyces cerevisiae brewer's yeast. The strains were characterized for their sugar utilization, phenolic off-flavors, hop sensitivity and flocculation. Trial fermentations were analyzed for extract reduction, ethanol formation, pH drop and final beers were analyzed for amino acids utilization and fermentation by-products. The performance of non-Saccharomyces strains and the commercial AFB strain were comparable during fermentation and production of fermentation by-products. An experienced sensory panel could not discriminate between the non-Saccharomyces AFB and the one produced with the commercial AFB strain, therefore indicating their suitability in AFB brewing.