The objective of our study was to evaluate the in vitro activity of levofloxacin in comparison with other antibiotics (coamoxiclav, ceftriaxone, ceftazidime, meropenem, aztreonam, ciprofloxacin and netilmicin) against about 500 Gram-negative nosocomial microorganisms, isolated during 2003. The bactericidal activities (minimal bactericidal activity and killing curve) of levofloxacin and the other antibiotics were also evaluated. The results confirm the excellent in vitro activity of levofloxacin against multiresistant nosocomial Gram-negative pathogens, including the 73 extended spectrum β-lactamase producer strains (90% of Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae were inhibited at 0.5 mg/l). Of particular interest is its susceptibility and bactericidal activity against Stenotrophomonas maltophilia strains. In conclusion, due to the favorable pharmacokinetics and pharmacodynamics of the molecule, associated with its safety, levofloxacin may represent a valid therapeutic option in the treatment of severe Gram-negative nosocomial infections.