Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.