Antibiotic resistance in bacteria is a major global threat and a leading cause for healthcare-related morbidity and mortality. Resistant biofilm infections are particularly difficult to treat owing to the protective biofilm matrix, which decreases both antibiotic efficacy and clearance by the host. Novel antimicrobial agents that are capable of eradicating resistant infections are greatly needed to combat the rise of antibiotic-resistant bacteria, particularly in patients with cystic fibrosis who are frequently colonized by multidrug-resistant species. Our research group has developed nitric oxide-releasing biopolymers as alternatives to conventional antibiotics. Here, we show that nitric oxide acts as a broad-spectrum antibacterial agent while also improving the efficacy of conventional antibiotics when delivered sequentially. Alone, nitric oxide kills a broad range of bacteria in planktonic and biofilm form without engendering resistance. In combination with conventional antibiotics, nitric oxide increases bacterial susceptibility to multiple classes of antibiotics and slows the development of antibiotic resistance. We anticipate that the use of nitric oxide in combination with antibiotics may improve the outcome of patients with refractory infections, particularly those that are multidrug-resistant.