Native microorganisms present on grapes can influence final wine quality. Chambourcin is the most abundant hybrid grape grown in Pennsylvania and is more resistant to cold temperatures and fungal diseases compared to Vitis vinifera. Here, non-Saccharomyces yeasts were isolated from spontaneously fermenting Chambourcin must from three regional vineyards. Using cultured-based methods and ITS sequencing, Hanseniaspora and Pichia spp. were the most dominant genus out of 29 fungal species identified. Five strains of Hanseniaspora uvarum, H. opuntiae, Pichia kluyveri, P. kudriavzevii, and Aureobasidium pullulans were characterized for the ability to tolerate sulfite and ethanol. Hanseniaspora opuntiae PSWCC64 and P. kudriavzevii PSWCC102 can tolerate 8–10% ethanol and were able to utilize 60–80% sugars during fermentation. Laboratory scale fermentations of candidate strain into sterile Chambourcin juice allowed for analyzing compounds associated with wine flavor. Nine nonvolatile compounds were conserved in inoculated fermentations. In contrast, Hanseniaspora strains PSWCC64 and PSWCC70 were positively correlated with 2-heptanol and ionone associated to fruity and floral odor and P. kudriazevii PSWCC102 was positively correlated with a group of esters and acetals associated to fruity and herbaceous aroma. Microbial and chemical characterization of non-Saccharomyces yeasts presents an exciting approach to enhance flavor complexity and regionality of hybrid wines.