The entrapment of fluoroquinolones, perfloxacine mesilate (PFX) and ofloxacin (OFX), in polyalkylcyanoacrylate (PECA) nanoparticles could offer some advantages for their biological application; for examples, increasing their bioavailability, controlling the drug time-release in blood, and reducing the formation of bacterial resistance. To load these two drugs in PECA polymeric bulk, the incorporation or adsorption method was performed. These two methods were capable of influencing nanoparticle size, molecular weight, release profile, and drug-polymer association. The incorporation method, particularly for the OFX system, achieved PECA nanoparticle suspensions with a mean size value three times higher than that obtained in the absence of the drug. In contrast, negligible changes were observed for PFX systems. This preparation process also influenced the nanoparticle storage stability. The molecular weight values of the various nanoparticle preparations were also influenced; that is, the PFX-loaded systems showed an enhancement in the average molecular weight values, whereas a reduction was observed for OFX-loaded systems. The adsorption method showed no particular difference in particle size, molecular weight, and storage stability compared with nanoparticles prepared without the drugs. The nanoparticle loading capacity was higher for the colloidal systems obtained following the incorporation preparation procedure. The release of drug from the nanoparticles was biphasic for both preparation processes. The fluoro-quinolone-loaded nanoparticles showed an enhancement of the antimicrobial activity against standard bacteria strains from 2- to 50-fold compared with the free drugs.