The capacity to form a biofilm was evaluated in Pseudomonas aeruginosa isolated from patients with lung and urinary infections. Adherence, development of microcolonies and slime formation varied in the studied strains. P. aeruginosa P63 isolated from cystic fibrosis (CF) exhibited important microcolony formation with the densest biofilm, and was selected to study the oxidative stress produced with ceftazidime and piperacillin by means of chemiluminescence (CL) in cell suspensions and biofilm. P. aeruginosa strain P63 was compared with P69; both were sensitive to ceftazidime and showed increase of reactive species of oxygen (ROS) in the presence of this antibiotic. P. aeruginosas P69 exhibited resistance to piperacillin and low ROS production, while piperacillin-sensitive strain P63 showed high oxidative stress with this antibiotic. Piperacillin stimulated oxidative stress, increasing ROS production only in the sensitive strain. Higher antibiotic concentrations were necessary to augment ROS in bacteria biofilm than in suspension. Incubation of P63 strain with ceftazidime or piperacillin in the presence of its own extracellular matrix (EM) or sodium alginate stimulated lesser oxidative stress and slower decrease of ROS than in the absence of these polysaccharides. A variant, V(10), obtained from strain P63 showed more sensitivity to the antibiotics than the wild-type, and concomitantly exhibited higher production of ROS in the presence of both the antibiotics studied.