A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' scores greater than 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole-cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2000 known bioactive compounds, 13 compounds generated reproducible signals >50% of that of the control (ciprofloxacin at one-half MIC) using bioluminescence readings after 7 h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other 3 are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis but by a variety of different DNA-damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA-damaging agents, which may lead to potent new antibacterials.