The aim of the present study was to examine the impact of macrolides on the expression of virulence factors and QS-associated genes in clinical P. aeruginosa isolates. Among 60 clinical P. aeruginosa , pyocyanin production was detected in 27 (45%) isolates, which belonged to various STs. Erythromycin inhibited the production of pigments in 12 out of 27 isolates. Other antibiotic categories didn't have an impact on production of pigments. Additionally, results showed that erythromycin sub-MIC inhibited the growth-rate in 17 isolates. Of note, in six isolates, the inhibition of growth-rate was greater when using both erythromycin and meropenem than using each antibiotic individually. Finally, addition of erythromycin down-regulated the expression of QS-associated genes (65.5%-81.3%) and almost all virulence-associated genes. In conclusion, our results confirmed that macrolides could be used in combination with last-line antibiotics, such as carbapenems, to treat infections caused by multidrug-resistant Gram-negative bacteria.