Kombucha beverage is a traditional and popular natural fermented beverage consumed across the globe. Kombucha beverage is produced by fermenting sweetened black tea, sucrose with consortium of acetic acid bacteria and yeasts. It is important to understand the microbial composition in Kombucha to facilitate better control of the fermentation process. Therefore, this study characterized the dominant acetic acid bacteria in Kombucha sample. Acetic acid bacteria isolated from the Kombucha using glucose-ethanol medium. Based on morphological, biochemical characterization, and 16S rRNA gene, 14 isolated strains belong to 2 species and were identified as Komagataeibacter saccharivorans and Komagataeibacter rhaeticus. These strains showed good biofilm forming abilities. Furthermore, the antimicrobial activity against five pathogenic bacteria including Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Samonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, Bacillus cereus ATCC 14579 of isolated acetic acid bacteria strains was also determined.