Inhibitors of the enzymes involved in fatty acid biosynthesis (FAB) have been reported as antibacterial agents. These include thiolactomycin, cerulenin, triclosan, diazoborine, naphthyridinones, aminopyridines and pyridoindoles. Our search for new FAB inhibitors, using a lacZ reporter cell-based screen, led to several confirmed hits.C ulture F92S91, later identified as a Pseudomonas sp. based on 16S profiling, was found to produce two α-pyrones (I and II) and three high molecular weight peptides. The pyrones were unstable under acidic conditions, and they were rearranged into a furanone derivative (III). Of these compounds, pyrone I was the most active with MICs (μg/ml) against B. subtilis (1-2), MRSA (2-4), M. catarrhalis (4) and VRE (2-64 . Effects on macromolecular synthesis and membrane functions were tested in B. subtilis. Pyrone I nonspecifically inhibited incorporation of radiolabeled precursors into DNA, RNA and protein within 5 minutes of drug exposure, similar to that of triclosan. Both compounds also inhibited the cellular uptake of these precursors. Cerulenin did not have an effect until 30 minutes of drug treatment. Pyrone I and triclosan were membrane-active (BacLight test); however, pyrone I (at ≤ 128 μg/ml concentration) was not hemolytic to human RBCs in contrast to triclosan, which was hemolytic at 16 μg/ml. These data suggest that pyrone-1, unlike triclosan, selectively affects bacterial membrane function.