Bacterial enoyl-ACP reductase (FabI) has been demonstrated to be a novel antibacterial target. In the course of our screening for FabI inhibitors, we isolated two methyl-branched fatty acids from Streptomyces sp. A251. They were identified as 14-methyl-9(Z)-pentadecenoic acid and 15-methyl-9(Z)-hexadecenoic acid by MS and NMR spectral data. These compounds inhibited Staphylococcus aureus FabI with $IC_{50}$ values of 16.0 and $16.3;{mu}M$, respectively, but did not affect FabK, an enoyl-ACP reductase of Streptococcus pneumonia, at $100;{mu}M$. Consistent with their selective inhibition for FabI, they blocked intracellular fatty acid synthesis as well as the growth of S. aureus, but did not inhibit the growth of S. pneumonia. Additionally, these compounds showed reduced antibacterial activity against fabI-overexpressing S. aureus, compared with the wild-type strain. These results demonstrate that the methylbranched fatty acids show antibacterial activity by inhibiting FabI in vivo.