Nowadays, antibiotic abuse increases the emergence of multidrug-resistant bacterial strains, which is the major reason for the failure of conventional antibiotic therapies. Therefore, developing novel antibacterial materials or therapies is an urgent demand. In the present study, photothermal and NOreleasing properties are integrated into a single nanocomposite to realize more efficient bactericidal effects. To this end, polydopamine (PDA) coated iron oxide nanocomposite (Fe 3 O 4 @PDA) is used as a photoconversion agent and the core, first three generation dendritic poly(amidoamine) (PAMAM-G3) is grafted on the surface of Fe 3 O 4 @PDA, and subsequently NO is loaded with the formation of NONOate. The resultant Fe 3 O 4 @PDA@PAMAM@NONOate displays controllable NO release property under intermittent 808 nm laser irradiation and excellent bacteria-separation efficiency. Moreover, excellent synergistic photothermal and NO antibacterial effects are observed against both Gramnegative Escherichia coli and Gram-positive Staphylococcus aureus, where bacterial viability and biofilm are significantly reduced. An antibacterial mechanism study reveals that the materials first adsorb onto the bacterial membrane, then cause damage to the membrane by the increased local temperature and the released NO under laser irradiation conditions, finally leak the intracellular components like DNA and induce bacteria death. The work provides a novel way for designing of antibacterial materials with higher efficiency.