Pseudomonas aeruginosa exhibits the biofilm mode of growth and causes chronic as well as acute infections in humans. Several reports have shown that the treatments with sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents influence biofilm formation by P. aeruginosa. The antibiotic ceftazidime (CAZ) is used to treat P. aeruginosa infections, but few studies have examined the effects of β-lactams on biofilm formation by P. aeruginosa. In this study, we investigated the role of sub-MICs of CAZ in the formation of P. aeruginosa biofilms. 1/4 × MIC CAZ reduced the biofilm volume of P. aeruginosa PAO1, as quantified by crystal violet staining. The formation of P. aeruginosa PAO1 biofilms treated with 1/4 × MIC CAZ were observed by confocal laser scanning microscopy. They were more heterogeneous than the PAO1 biofilms without CAZ treatment. Furthermore, sub-MICs of CAZ inhibited the twitching motility, which played an important role in mature biofilm formation. 1/4 × MIC CAZ also reduced the gene expressions of lecA, lecB, pel and psl, which mediate the adhesion and polysaccharide matrix synthesis of P. aeruginosa. These effects suggest that sub-MICs of CAZ may affect a number of stages of biofilm formation. Investigating the effects of sub-MIC antibiotics on targeted bacterial biofilm may lead to the development of future antibiotic treatment modalities.