AbstractBiofilms are currently responsible for 80% of human chronic bacterial infections, being composed of bacterial communities within self‐produced extracellular polymeric substances (EPS) that can resist various adverse factors in the bacterial microenvironment. Therefore, the development of powerful antibacterial systems by disrupting biofilms first and killing exposed free‐living bacteria is the top priority for clinical antibiotic needs. In this study, we developed a pH‐ and photothermally responsive photosensitizer/enzyme‐loaded nanocomposite for enhanced biofilm disruption and bacteria killing (gram‐positive and gram negative). To achieve this, IR780 (I) as an efficient NIR dye was encapsulated inside the hydrophobic core of 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycerol)‐2000] (DSPE‐PEG2000) micelles (M) for enhanced photothermal therapy. EPS extracellular DNA lyase deoxyribonuclease I (DNase I) was anchored on the micellar surface by calcium phosphate mineralization method. The results indicated that pH‐sensitive MI@CaPD nanocomposite degraded as a response to the acidic conditions characteristic for the bacterial environment and released DNase I and Ca2+ ions simultaneously. Subsequently, the Ca2+ stabilized the DNase I active structure and facilitated the dispersion of the biofilm EPS. Then, interior bacteria were exposed and killed by IR780‐mediated hyperthermia. The synergistic effect of DNase I and photothermal therapy could efficiently eradicate the biofilms, which exhibits superior biofilm dispersion and destruction capability.