The rise in microbreweries has resulted in a significant increase in craft beer production. This has created a need for new and innovative brewing methods to compete in the market and meet consumers’ demands. One of these innovative methods is using wild yeasts, also known as “non-conventional” yeasts, to enhance the flavor and aroma of various beer styles. With its diverse ecosystems and rich culinary culture, Ecuador has a long history of preparing fermented foods and beverages, providing a natural source of wild yeasts. This study evaluated the fermentation behavior of two strains of S. cerevisiae, isolated from a traditional fermented beverage called chicha, to identify optimal conditions for controlled fermentation and brewing a Pale Ale-style craft beer. The two “non-conventional” strains were compared with a control yeast (SafAle S-04) by measuring parameters during fermentation and conducting sensory evaluations of the brewed beers. The pure cultures of the two strains (ScCh and ScA) and the mixed culture (ScCh:ScA) all successfully fermented the barley wort and produced beers with profiles similar to those of the control strain (ScC). Due to the high biomass production, they also exhibited good, pronounced fermentative flavors and aromas. The optimal growth temperature was 20 ºC, although growth was also observed at 12 ºC, but at a slower rate. Co-inoculation (ScCh:ScA) demonstrated no advantages over pure strains in the tests. Two evaluated S. cerevisiae strains show potential for innovation, differentiation, and diversification of craft beer. However, further study of their fermentative potential and enzymatic activities is necessary.