FabH and FabF are essential enzymes in type II fatty acid synthesis and are promising targets for antibacterial drug discovery and development. A new approach using a xylose inducible plasmid to express antisense RNA (AS-RNA) in Staphylococcus aureus has been recently described. In order to identify FabF/FabH target specific cell permeable inhibitors from natural products, we developed an agar-diffusion two-plate differential sensitivity assay. Because both the fabH and fabF genes share the same operon, the increase in fabF AS-RNA levels decreases the expression of FabH and FabF proteins, making the cells more sensitive to FabF and/or FabH inhibitors. Using this assay, we screened over 250,000 natural product extracts followed by confirmation in biochemical assays, giving a hit rate of 0.1%. We discovered all known FabH and FabF inhibitors that included cerulenin, thiolactomycin, thiotetromycin and Tu3010 from natural product extracts for the first time using a mechanism based screening approach. We discovered a number of novel natural products as FabF inhibitors including platensimycin and platencin. The details of discovery process, structures, biological activities, in vivo efficacy, mechanism of action and inhibitor bound X-ray crystal structure will be discussed.