Non-Saccharomyces wine yeasts are recognized for their ability to enhance the aroma complexity of wines. New trends in winemaking have highlighted the concept of microbiological terroir thus encouraging the use of indigenous non-Saccharomyces starter cultures in mixed fermentations with Saccharomyces cerevisiae to overcome the lack of authenticity and provide the wines with unique regional character and distinct aromatic profiles. In this study, 257 indigenous non-Saccharomyces isolates were obtained for the first time from 11 Maraština vineyards located within the winegrowing region of Dalmatia (Croatia). The isolates were identified at the molecular level and characterized for desirable oenological traits such as growth at different temperatures, high ethanol and SO2 tolerance, and for negative features such as high hydrogen sulphide (H2S) and acetic acid production as well as hydroxycinnamic acid decarboxylase (HCDC) activity. Among 27 identified species, those from Aureobasidium genera prevailed, followed by Metschnikowia, Hanseniaspora and Lachancea. The representatives from different species showed high SO2 (up to 250 mg/L) and ethanol (up to 12%) tolerance, low acetic acid, and medium-low H2S production. The HCDC activity was detected exclusively among Metschnikowia, Aureobasidium and Kabatiella species. Some regionally distinct characteristics within the isolates from the same species were observed. The encouraging insights into potential new starter cultures emerged after the evaluation of enzymatic activities of selected isolates. Most of them showed high leucine and valine arylamidase activity, whereas Hanseniaspora and Metschnikowia isolates were additionally characterized by high β-glucosidase and esterase activity, respectively. Display omitted •Indigenous non-Saccharomyces yeasts were isolated from Croatian Maraština grapes.•Among 257 isolates, 27 different species were identified and characterized.•Potential candidates for development as starter cultures were found.•Hanseniaspora isolates showed high β-glucosidase activity.•Some regionally distinct characteristics within the species were observed.