Due to its ubiquity, ability to form biofilms, and acquire resistance mechanisms, Pseudomonas spp. become one of the major challenge for healthcare settings and food industry. The aims of this study were to assess the biofilm production of Pseudomonas spp. recovered from clinical and food specimens and to evaluate their antimicrobial resistance. A total of 108 isolates of Pseudomonas spp. were included in the study, 48 being clinical isolates recovered from patients admitted to four tertiary care hospitals throughout Serbia and 60 were isolated from the bulk tank milk samples and meat carcasses. Biofilm production was analyzed by microtiter plate assay. Antimicrobial susceptibility was evaluated by disk diffusion method according to the CLSI guidelines, while class A and B beta-lactamases encoding genes were screened by PCR. A total of 98 (90.7%) strains were biofilm producers (moderate producer: 68, 69.4%; strong producer: 8, 8.2%). Although a slightly higher percentage of clinical isolates were biofilm producers (91.7%) compared to food isolates (90%), statistical significance was not observed (P > 0.05). The proportion of carbapenem-resistantPseudomonas aeruginosa(CRPA) isolates was significantly higher among clinical (42%) isolates compared to food (1.7%) Pseudomonads (P < 0.05). Thebla(PER)and bla(NDM)genes were found in ESBL (seven isolates) and MBL (two isolates) production, respectively. In the present study, we confirmed that biofilm formation was highly present in both clinical and foodPseudomonasspp. irrespective of the prior existence of resistance genes. Additionally, clinical settings pose a major reservoir of MDRPseudomonasspp. and especially CRPA isolates.