Introduction: Polymeric nanoparticles are promising nanotechnology tools to fight pathogenic bacteria resistant to conventional antibiotics. Objective: To synthesize polylactic acid nanoparticles loaded with ofloxacin and vancomycin, and to determine their antibacterial activity against Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA). Materials and methods: We synthesized ofloxacin or vancomycin loaded polylactic acid nanoparticles by the emulsification-solvent evaporation method, and characterized them by dynamic light scattering, laser Doppler electrophoresis and scanning electron microscopy. We evaluated in vitro antibacterial activity of ofloxacin- and vancomycin-loaded polylactic acid nanoparticles against E. coli O157:H7 and MRSA using the broth microdilution method. Results: Ofloxacin- and vancomycin-loaded polylactic acid nanoparticles registered a positive surface charge density of 21 mV and an average size lower than 379 nm. In vitro minimum inhibitory concentration (MIC50) of ofloxacin-polylactic acid nanoparticles was 0,001 mu g/ml against E. coil O157:H7, i.e., 40 times lower than the free ofloxacin (MIC50: 0.04 mu g/ml), indicating enhanced antibacterial activity while the in vitro MIC50 of vancomycin-polylactic acid nanoparticles was 0,005 mu g/ml against MRSA, i.e., 100 times lower than that of free vancomycin (MIC50: 0.5 mu g/ml). Conclusion: Polylactic acid nanoparticles loaded with ofloxacin and vancomycin showed a higher antibacterial activity. Polymeric nanoparticles are a possible alternative for drug design against pathogenic bacterial strains of public health interest.