This study evaluated the in vitro probiotic potential of 15 yeast strains isolated from commercial kombucha samples in New Zealand. The strains were assessed for their growth under various conditions, including low pH, bile salts, and different temperatures and salt concentrations. The cell surface characteristics, such as auto-aggregation, co-aggregation, and hydrophobicity, were also examined. Additionally, the strains were tested for their antioxidant, antimicrobial, and bile salt hydrolase (BSH) activities. The results showed that six strains (Dekkera bruxellensis LBY1, Saccharosacharomyces pombe LBV5, Hanseniaspora walyensis DOY1, Bretanomyces anomalus DOY8, Pichia kudriavzevii GBY1, and Saccharomyces cerevisiae GBY2) exhibited good growth under low-acid conditions and in the presence of bile salts, suggesting their potential to survive in the human gut. All strains showed negative enzymatic activity reactions, indicating safety for consumption. Notably, two strains (Pichia kudriavzevii GBY1 and Saccharomyces cerevisiae GBY2) demonstrated desirable cell surface hydrophobicity, auto-aggregation, co-aggregation, resistance to eight tested antibiotics, and high antioxidant activities. These findings suggest that these two strains have potential probiotic activities and could be useful in functional food production.This study evaluated the in vitro probiotic potential of 15 yeast strains isolated from commercial kombucha samples in New Zealand. The strains were assessed for their growth under various conditions, including low pH, bile salts, and different temperatures and salt concentrations. The cell surface characteristics, such as auto-aggregation, co-aggregation, and hydrophobicity, were also examined. Additionally, the strains were tested for their antioxidant, antimicrobial, and bile salt hydrolase (BSH) activities. The results showed that six strains (Dekkera bruxellensis LBY1, Saccharosacharomyces pombe LBV5, Hanseniaspora walyensis DOY1, Bretanomyces anomalus DOY8, Pichia kudriavzevii GBY1, and Saccharomyces cerevisiae GBY2) exhibited good growth under low-acid conditions and in the presence of bile salts, suggesting their potential to survive in the human gut. All strains showed negative enzymatic activity reactions, indicating safety for consumption. Notably, two strains (Pichia kudriavzevii GBY1 and Saccharomyces cerevisiae GBY2) demonstrated desirable cell surface hydrophobicity, auto-aggregation, co-aggregation, resistance to eight tested antibiotics, and high antioxidant activities. These findings suggest that these two strains have potential probiotic activities and could be useful in functional food production.