Bacterial protein synthesis inhibitors interact mainly with rRNA and to some extent ribosomal proteins, which are potential targets for developing new antibacterial agents. Specifically, the ribosomal protein S4 of the 30s ribosomal subunit known as ribosomal protein small-subunit D (rpsD) may be useful as a target. The antisense-rpsD gene-sensitized two-plate assay led to the discovery of a novel chlorinated cyclopentandienylbenzopyrone antibiotic, coniothyrione, C14H9ClO6, isolated from Coniothyrium cerealis MF7209. It exhibited liquid MICs of 16-32 microg/mL against Staphylococcus aureus, Bacillus subtilis, Haemophilus influenzae, Streptococcus pneumoniae, and Enterococcus faecalis and >64 microg/mL against Escherichia coli. Isolation, structure elucidation, and antibacterial activity of coniothyrione are described.