BackgroundEmergence of multi-drug resistantPseudomonas aeruginosa,coupled with the pathogen’s versatile virulence factors, lead to high morbidity and mortality rates. The current study investigated the potential association between the antibiotic resistance and the production of virulence factors amongP. aeruginosaclinical isolates collected from Alexandria Main University Hospital in Egypt. We also evaluated the potential of the phenotypic detection of virulence factors to reflect virulence as detected by virulence genes presence. The role of alginate in the formation of biofilms and the effect of ambroxol, a mucolytic agent, on the inhibition of biofilm formation were investigated.ResultsA multi-drug resistant phenotype was detected among 79.8% of the isolates. The most predominant virulence factor was biofilm formation (89.4%), while DNase was least detected (10.6%). Pigment production was significantly associated with ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to cefepime, and DNase production was significantly associated with intermediate resistance to meropenem. Among the tested virulence genes,lasBandalgDshowed the highest prevalence rates (93.3% and 91.3%, respectively), whiletoxAandplcNwere the least detected ones (46.2% and 53.8%, respectively). Significant association oftoxAwith ceftazidime susceptibility,exoSwith ceftazidime and aztreonam susceptibility, andplcHwith piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection ofalgD,lasB,exoS,plcHandplcN; pigment production and the presence ofalgD,lasB,toxAandexoS; and gelatinase production and the existence oflasB,exoSandplcH. Ambroxol showed a high anti-biofilm activity (5% to 92%). Quantitative reverse transcriptase polymerase chain reaction showed that alginate was not an essential matrix component inP. aeruginosabiofilms.ConclusionsHigh virulence coupled with the isolates’ multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates amongP. aeruginosainfections. Ambroxol that displayed anti-biofilm action could be suggested as an alternative treatment option, yet in vivo studies are required to confirm these findings. We recommend active surveillance of antimicrobial resistance and virulence determinant prevalence for better understanding of coregulatory mechanisms.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02897-8.