In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents. One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes. We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B. subtilis chromosome. Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials. Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds. The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin). Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening. In summary, we successfully generated B. subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials. The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner.