Lactic acid bacteria (LAB) effects are related to producing anti-microbial agents as bacteriocin. Our target is to isolate LAB from different horse milk samples and investigates its antimicrobial activities against many pathogenic bacteria. LAB was isolated from horse milk from Al-Taif city in Saudi Arabia on MRS medium. By agar well diffusion method, twelve LAB isolates demonstrated that the antibacterial activities against Gram-positive bacteria S. aureus ATCCBAA977, ST. pneumonia ATCC49619, and E. coli ATCC35218, P. aeruginosa ATCC27853. According to the 16S rDNA sequence, the highest activity LAB isolate was presented in a gene bank such as Lactobacillus plantarum with an identity of 99% under accessory LBP 135401.1. The results showed that the incubation at 35 ° C for 24h at pH 6.5 were the best conditions for producing the largest quantity of bacteriocin.