Lactic acid bacteria (LAB) can be isolated from different natural sources such as plants, fruits, or vegetables. The organic acid-producing capacity and antibacterial activity of each LAB strain are highly dependent on its isolation source. In the study, LAB strains were isolated from guava (Psidium guajava L.), identified for organic acid production and antibacterial activity. Twenty LAB isolates showed rod, oval, or cocci shape, Gram-positive, catalase-negative, oxidase-negative, the ability to degrade CaCO₃, and could not produce indole from tryptophan. Especially, the strain TN2 produced the highest organic acid content (8.7±0.14 g/L) as well as exhibited the strongest antibacterial activity against Escherichia coli (E. coli) with the inhibition zone diameter of 12.33±2.08 mm. The bacteria strain TN2 was applied to ferment yogurt with 8% guava juice, at a milk and condensed milk ratio of 2:1, giving the best lactic acid content and sensory evaluation. The strain TN2 was identified as Lactococcus lactis strain according to the 16S rRNA gene sequence analysis and registered on GenBank with code number MN860000.1. This strain can be a potent alternative to commercialized LAB strains in fermented food products.