Acetic acid bacteria strains were isolated from commercial natural vinegar. Ten isolated strains were evaluated for ethanol- and sulfur-tolerance and identified using 16S rRNA gene sequencing data. Eight strains of isolated strains were identified as Acetobacter pasteurianus. A. pasteurianus JAC002, JAC005 and JAC008 strains showed a high ethanol tolerance, resulting in selecting for manufactureing “Makgeolli seed-vinegar”. Makgeolli seed-vinegars were manufactured using selected strains, and their physicochemical properties, free sugars, organic acids, and antimicrobial activities were evaluated. A. pasteurianus JAC002 strain showed the highest acetic acid production ability on the twentieth day of fermentation. It’s total acidity, acetic acid production content, and ethanol consumption content were 4.21%, 3775.06 mg%, and 2931.78 mg%, respectively. As the acetic acid content of JAC002 increased, the antimicrobial activities were increased. As a result, A. pasteurianus JAC002 strain was evaluated as the most suitable strain for seed-vinegar. Tomato vinegar were manufactued using the Makgeolli seed-vinegar, and their physicochemical properties, antioxidant, antimicrobial, anti-obesity, anti-inflammatory activities were evaluated. Total acidity of tomato vinegar was increased by more than 4% on the twentieth day of fermentation. The lycopene content, DPPH and ABTS·+ radical scavenging activity of tomato vinegar were higher than control(tomato wine), but the content and activity decreased with increasing amount of seed-vinegar. Antimicrobial and anti-inflammatory activities were improved with increasing amount of seed-vinegar, but the difference was small. In the anti-obesity activity, it was confirmed that lipid droplet formation was inhibited in all experimental groups, but activity was decreased with increasing amount of seed-vinegar. As a result, A. pasteurianus JAC002 strain is considered to be useful as a starter strain of seed-vinegar. And it is considered that 20% of the addition of seed-vinegar is more useful than more of the addition of seed-vinegar for various useful substances and functionalities of fruit.