The spread of multidrug-resistant (MDR) bacteria poses a great risk to global health, and despite considerable efforts, no new class of antibiotic has been approved for Gram-negative pathogens in recent years. Arylomycins are a class of natural products that exhibit low antibacterial activity. By chemically modifying arylomycin, the authors discovered a synthetic derivative, termed G0775, with potent in vitro and in vivo antibacterial activity against multiple clinically relevant MDR Gram-negative pathogens. G0775 binds to the bacterial signal peptidase LepB, a new antibiotic target, with high affinity and kills bacteria by inhibiting this peptidase. Moreover, G0775 has improved ability to cross the outer membrane through a porin-independent mechanism, and de novo resistance to G0775 occurred at a low frequency. Thus, optimized arylomycin analogues may represent a new class of Gram-negative antibiotics.