Water kefir is the product of a brown sugar solution fermented with water kefir grains. To produce starter cultures for manufacturing commercial water kefir on an industrial scale, six lactic acid bacteria (LAB), three acetic acid bacteria (AAB), and two yeast strains have been isolated from local water kefir grains, and in vitro α-glucosidase inhibitory activity has been investigated. Five of the 11 isolates (LAB [K65, K9, K67], AAB [A5], and yeast [Y6]) showed high α-glucosidase inhibitory activity. They were then tested in an in vitro gastrointestinal tract tolerance test in which all demonstrated tolerance (40‒80% survival rate). These five strains, identified as Lactobacillus mali, Lactobacillus casei, Leuconostoc mesenteroides, Gluconobacter hansenii and Saccharomyces cerevisiae; were then used to ferment water kefir beverages. The storage stability of the fermented beverage products was analysed during 28 days of storage at 4°C by measuring α-glucosidase inhibitory activity, microbial cell viability, and microbial survival in the beverage treated with simulated gastric juice. The water kefir beverage sample containing both pumpkin purée and brown sugar fermented using all five strains (109 CFU/mL LAB strains, 107 CFU/mL yeast strain, and 106 CFU/mL AAB strain) was the most suitable formulation because it achieved >50% α-glucosidase inhibitory activity and microbial survival rates of 106‒1010 CFU/mL viable LAB cells, 106‒107 CFU/mL viable AAB cells, and 105‒108 CFU/mL viable yeast cells during storage. Therefore, these five strains have potential to act as starters in the formulation of water kefir, which is an anti-hyperglycaemic beverage that can be used to manage type-2 diabetes mellitus.