Biocompatible Fe3O4/chitosan (CS)/poly (ethylene glycol) (PEG)/gentamicin (Gent) magnetic nanoparticles, namely Fe3O4@PEG-Gent NPs, have been successfully developed for antibiotic delivery. In which, PEG dicarboxylic acid was used to modify Fe3O4 NPs for good dispersity as well as offer sufficient carboxyl groups as binding sites. And then the free Gent was facilely loaded onto Fe3O4 NPs so as to achieve powerful antibacterial activity via electrostatic interactions. Under acidic condition, the CS and PEG of Fe3O4@PEG-Gent were protonated to introduce the positive charge to NPs surface, thus facilitating the contact with negatively charged bacterial cell membrane. What is more, the stretches of CS chains triggered by acidic pH may prevent the antimicrobial efficiency of Gent from weakening. Compared with the free antibiotic, these nanocomposites presented better antimicrobial efficacy against gram-positive bacteria S. aureus under acidic condition. Intriguingly, the confocal laser scanning macroscopy imaging suggested that the anti-biofilm efficacy of nanocomposites was significantly enhanced in the presence of an external magnetic field. Due to the superparamagnetic performance of Fe3O4 NPs, these nanocomposites were allowed deeper penetration into a mature biofilm of S. aureus by magnetic field, leading to an effective Gent delivery for eradication of biofilm. The ingenious fabrication of the antibiotic delivery system not only efficiently improved the effectiveness and bioavailability of Gent at acidic media, but also provided an innovative platform to treat bacterial biofilms-associated infection by applying extra environmental factors such as magnetic field.