There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs. Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV and HCV. NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites could limit resistance emergence. Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the "i+1" NTP binding site) and mechanism (competition with UTP for occupancy of the "i+1" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.