The risk of emergence and spread of microbial pathogens increased due to the overuse of sterilization and disinfection materials with a lack of health supervision. We aimed to determine the extent of sensitivity of P.aeruginosa to biocides regularly used in the hospitals, and to study synergistic effect of (EDTA) with the same biocides. Twenty five isolates of P.aeruginosa were diagnosed, from 200 samples. (100 clinical and 100 environmental) samples were collected from Al-Diwaniyah Teaching Hospital. Samples were cultivated on blood agar, macconkey agar and cetrimide agar, and bacterial isolates were identified according to Hemolytic lysis of the blood cells in blood agar, unablity to ferment the sucrose and lactose on macconkey agar and the blue greenish pyocyanine stain and odour as fruit on cetrimide agar. Isolates were confirmed using the VITEK2 automated system. A standard broth dilution method was used to determine the MIC concentrations of chlorhexidine digluconate (20%), benzalkonium chloride (50%), formaldehyde (37%), cetrimide (20%), and hydrogen peroxide H2O2 (30 %) at concentrations ranging from 4 µg/ml to 2048 µg/ml. 17% EDTA was used for synergistic test. The results showed that the MIC ranges for the biocides used were as follows: chlorhexidine digluconate (64 - ≥2048 µg/ml) (8-64 µg/ml), formaldehyde (64 -≥1024 µg/ml) (16 - ≥128 µg/ml), cetrimide (256 - ≥2048 µg/ml) (4 - ≥256 µg/ml), benzalkonium chloride (512 - ≥2048 µg/ml) (8 - ≥256 µg/ml), and hydrogen peroxide (≥2048 µg/ml) (4 - ≥256 µg/ml) without and with EDTA respectively. The MBC concentrations were: chlorhexidine digluconate (128-≥2048 μg/ml), benzalkonium chloride (1024-≥2048 μg/ml), formaldehyde (128-≥2048 μg/ml), cetrimide (1024-≥2048 μg/ml) and hydrogen peroxide (≥2048 μg/ml). A combination of biocides and EDTA has been recommended as an effective way to reduce hospital contamination by P.aeruginosa.