The dispersal phase that completes the biofilm lifecycle is of particular interest for its potential to remove recalcitrant, antimicrobial tolerant biofilm infections. Here we found that temperature is a cue for biofilm dispersal and a rise by 5 °C or more can induce the detachment of Pseudomonas aeruginosa biofilms. Temperature upshifts were found to decrease biofilm biomass and increase the number of viable freely suspended cells. The dispersal response appeared to involve the secondary messenger cyclic di-GMP, which is central to a genetic network governing motile to sessile transitions in bacteria. Furthermore, we used poly((oligo(ethylene glycol) methyl ether acrylate)-block-poly(monoacryloxy ethyl phosphate)-stabilized iron oxide nanoparticles (POEGA-b-PMAEP@IONPs) to induce local hyperthermia in established biofilms upon exposure to a magnetic field. POEGA-b-PMAEP@IONPs were non-toxic to bacteria and when heated induced the detachment of biofilm cells. Finally, combined treatments of POEGA-b-PMAEP@IONPs and the antibiotic gentamicin reduced by 2-log the number of colony-forming units in both biofilm and planktonic phases after 20 min, which represent a 3.2- and 4.1-fold increase in the efficacy against planktonic and biofilm cells, respectively, compared to gentamicin alone. The use of iron oxide nanoparticles to disperse biofilms may find broad applications across a range of clinical and industrial settings.