AbstractThe formation of bacterial biofilms on material surfaces is a recurrent problem in public health. Antibacterial nanoparticles (NPs) are promising because pathogens have not yet developed resistance mechanisms and encapsulation of the drug can protect it from the surrounding medium and improve pharmacokinetics. Biocompatible and biodegradable particles of various sizes (nano‐ and micro‐scale) based on poly(lactic‐co‐glycolic acid) (PLGA) are elaborated using a simple and free toxic nanoprecipitation process. Particles are poly(ethylene glycol) (PEG)‐ylated in order to reduce unwanted interactions with biological fluids, or loaded with the large spectrum antibiotic ciprofloxacin (CIP). NPs are studied against Staphylococcus aureus in planktonic and biofilm modes. Empty NPs with smallest size (60 nm) are able to totally eradicate planktonic culture after 24 h, even in the presence of serum proteins. CIP‐loaded NPs present slightly lower antimicrobial activity against planktonic microorganisms compared with the free antibiotic, due to progressive release of CIP over time. In biofilm mode, CIP‐loaded NPs show a very good antibiofilm activity, much better than free CIP, thanks to NPs penetration within the polymer matrix and a consequent release of the antibiotic close to the embedded bacteria. The present results open the way for widespread applications of PLGA‐NPs in the pharmaceutical or medical fields.