The present study aimed to characterize the susceptibility profile of Pseudomonas aeruginosa and Acinetobacter spp. clinical isolates to polymyxin B in a public hospital in Recife-PE, Brazil, between the years of 2018 and 2019, as well as to search for the presence of the mcr-1 gene and evaluate the interaction between polymyxin B and usnic acid against these isolates. The strains were identified using the BD Phoenix™ automated system and the agar-spot test was used to determine the susceptibility profile to polymyxin B. The minimum inhibitory concentrations (MICs) of usnic acid and polymyxin B were determined through the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). Subsequently, Polymerase Chain Reaction (PCR) was performed to detect the mcr-1 gene in the isolates. The interaction between usnic acid and polymyxin B was evaluated by the Checkerboard assay. Among 34 isolates of P. aeruginosa, 26.5% (9/34) were positive for the polymyxin B agar-spot test, and 11.8% (4/34) presented an intermediate susceptibility (MIC = 4 μg/mL), while 14.7% (5/34) presented antimicrobial resistance with MIC values ranging from 8 to 32 μg/mL. Among 38 isolates of Acinetobacter spp., 13.2% (5/38) were positive for the polymyxin B agar-spot test and all of them were resistant to polymyxin B with a MIC value > 32 μg/mL. The mcr-1 gene was not detected in the clinical isolates. Regarding usnic acid, it presented a moderate antibacterial activity against two P. aeruginosa isolates (MIC = 250 μg/mL) and no activity was detected against the others. A synergistic effect between usnic acid and polymyxin B was observed against three clinical isolates of P. aeruginosa which were resistant to polymyxin B (FICI ≤ 0.5). Therefore, it was possible to observe that usnic acid is a promising candidate to be used in combination with polymyxin B against infections caused by resistant P. aeruginosa.