Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.