An attractive strategy for combating antibacterial resistance involves the development of new antibiotics whose mechanisms differ from those of existing ones in the clinic. Elfamycin antibiotics, whose prototypes include kirromycin and aurodox, are illustrative examples based on their ability to target EF-Tu, an essential component for protein translation in bacteria. Our efforts to revisit this antibiotic class were enabled by two developments. First, we produced L-681,217, an understudied member of this polyketide family harboring a terminal carboxylic acid in place of a hydroxypyridone ring, and synthesized a biotinylated derivative with comparable activity to the natural product. Second, we established a sensitive cell-free protein synthesis (CFPS) assay in which superfolder green fluorescent protein (sfGFP) production was inhibited by L-681,217. Biotinyl-L-681,217 was used to drain the CFPS system of endogenous EF-Tu, allowing replenishment with orthologs to interrogate pathogen selectivity and propensity toward resistance. Comparative in vitro analysis of kirromycin and L-681,217 showed that, while both antibiotics are equipotent in CFPS assays, they interact distinctly with purified EF-Tu, a feature that presumably correlates with prior observations that kirromycin enhances GTP hydrolysis by EF-Tu whereas L-681,217 does not. Analysis of L-681,217 and kirromycin accumulation in selected mutant E. coli strains also revealed that antibiotic import and efflux contributed to resistance. The promise of L-681,217 as a medicinal lead was underscored by the observation that, unlike aurodox, this polyketide does not inhibit adenylosuccinate synthase.