In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.