This paper describes the optimization of the preparation of ciprofloxacin-loaded polyethylbutylcyanoacrylate (PEBCA) nanoparticles. The association of ciprofloxacin with nanoparticles was performed by emulsion polymerization, but successful entrapment was only obtained in the presence of acetone in the polymerization medium. This preparation process led to a stable ciprofloxacin nanoparticle suspension, with a mean size value twice as high as that obtained in the absence of drug, and an association efficiency of 82%. Moreover, the molecular weight value of ciprofloxacin nanoparticles was shown to be reduced as compared with unloaded nanoparticles. Drug release from the colloidal carrier in medium containing esterase was found to be very slow (a maximum of 51.5% after 48 h), suggesting that this release resulted from bioerosion of the polymer matrix. Interestingly, it was observed that 30.5% of the initial amount of ciprofloxacin was not detectable by HPLC analysis after nanoparticle preparation and corresponded either to ciprofloxacin covalently bound to PEBCA or to ciprofloxacin chemically degraded during the polymerization process. 19F-NMR analysis demonstrated that ciprofloxacin entrapped into nanoparticles was only in its neutral form. The measurements of molecular weight suggest the participation of the antibiotic as an anionic polymerization initiator, leading to the formation of a chemical bond between some of the drug and the polymer. These data allowed us to propose a model describing the association of ciprofloxacin with PEBCA nanoparticles obtained by emulsion polymerization.