Constructing a new antibacterial structural framework is an effective strategy to combat drug resistance. This work discovered a class of naphthalimidopropanediols (NIOLs) as a novel structural type of potential broad-spectrum antibacterial agents. Especially, NIOLs 9u, 12i, 15 against Staphylococcus aureus and NIOLs 9l, 13a against Pseudomonas aeruginosa showed excellent inhibitory activities, and they displayed high membrane selectivity from an electrostatic distinction on the membranes between bacteria and mammalian cells. These highly active NIOLs could effectually inhibit the bacterial growths, and relieve the resistance developments. Moreover, the facts of membrane depolarization, outer/inner membrane permeabilization and leakage of intracellular materials, demonstrated that these NIOLs could target and destroy the S. aureus or P. aeruginosa membranes. In particular, they could disrupt the antioxidant defense systems of S. aureus or P. aeruginosa through up-regulation of reactive oxygen species. Simultaneously, they could render the metabolic inactivation of the tested strains, and eradicate the formed biofilms and efficiently kill the strains within the biofilms. The in vitro and in vivo cytotoxicity assay indicated that these compounds possessed low toxicity. These findings of novel NIOLs as potential broad-spectrum antibacterial members provided a bright hope for conquering drug resistance.