Background This study aimed to investigate the association between biocides' reduced susceptibility and the presence of efflux pump genes including cepA, qacE Delta 1 and qacE in multidrug-resistant (MDR) Pseudomonas aeruginosa. Methods and results The MDR P. aeruginosa isolates were collected and identified from different clinical samples. The minimum inhibitory concentrations (MIC) of four biocides (chlorhexidine gluconate 1%, benzalkonium chloride 1%, Kohrsolin (R) extra, and SEPTI-Turbo) were determined by microbroth dilution with and without carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Polymerase chain reaction (PCR) was performed for detecting the efflux pump genes. In total, 92 MDR P. aeruginosa isolates were collected. The reduced susceptibility (8-128 mu g/ml) was seen against chlorhexidine gluconate 1%, benzalkonium chloride 1%, Kohrsolin (R) extra, and SEPTI-Turbo in 63 (68.5%), 59 (64.1%), 64 (69.6%), and 65 (70.6%) isolates, respectively. The Kohrsolin (R) extra was the most effective biocide. The cepA, qacE, and qacE Delta 1 were detected in 56 (60.9%), 1 (1.1%), and 34 (36.9%) isolates, respectively. There was a significant association between the presence of biocide resistance genes and reduced susceptibility to studied biocides (P = 0.00001). The CCCP had no effect on benzalkonium chloride 1% and Kohrsolin (R) extra, but reduced the MICs of chlorhexidine gluconate 1% and SEPTI- Turbo by 2 to 128 fold. Conclusions The P. aeruginosa isolates exhibited varying degrees of tolerance to biocides. The cepA was the most prevalent gene. There was a significant connection between the occurrence of the efflux pump genes cepA and qacE Delta 1 with reduced biocide susceptibility.