The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) andAcinetobacter baumannii. For CRE, the MIC50and MIC90for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressingKlebsiella pneumoniaeNevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to theA.baumannii70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia colinumbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in singlerrnoperonE.coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.