The emergence of resistance to multiple antimicrobial agents by pathogenic bacteria has become a significant global public health threat. Multi-drug-resistant (MDR) Gram-negative bacteria have become particularly problematic, as no new classes of small-molecule antibiotics for Gram-negative bacteria have emerged in over two decades. We have developed a combinatorial screening process for identifying mixed ligand monolayer/gold nanoparticle conjugates (2.4 nm diameter) with antibiotic activity. The method previously led to the discovery of several conjugates with potent activity against the Gram-negative bacterium Escherichia coli. Here we show that these conjugates are also active against MDR E. coli and MDR Klebsiella pneumoniae. Moreover, we have shown that resistance to these nanoparticles develops significantly more slowly than to a commercial small-molecule drug. These results, combined with their relatively low toxicity to mammalian cells and biocompatibility in vivo, suggest that gold nanoparticles may be viable new candidates for the treatment of MDR Gram-negative bacterial infections.