Biofilm production is an important mechanism for bacterial survival and its occurrence together with antimicrobial resistance represents a challenge for clinical management. Here, we evaluated the ability for biofilm production among P. aeruginosa isolates from patients with or without cystic fibrosis (CF) using two distinct media, besides determining the antimicrobial susceptibility profile of these isolates for eight antimicrobial agents. The ability for biofilm production when TSB medium was used was higher than when used CF sputum media (p = 0.0198). However, P. aeruginosa isolates from CF have demonstrated similar performance for biofilm production, independently of the medium used. Besides, among the biofilm-producing isolates, those recovered from CF were more resistant to the carbapenems (meropenem and imipenem) agents than those isolates from non-CF isolates.