Antibiotic resistance has become one of the most common problems that threaten the world and increases the mortality rate. Pseudomonas aeruginosa is reported to be one of commonest Multi-Drug resistant (MDR), which is responsible for 10 to 15% of nosocomial infection worldwide and high death rates ranging from 18 to 61%. We investigate the anti-biofilm activity of different disinfectants on MDR Pseudomonas aeruginosa. Two hundred two water environmental isolates were collected from different hospitals in Cairo, Egypt. Microbact™ Gram-negative system used for identification. That is showed 41.3% of isolates were Pseudomonas aeruginosa. Antibiotic susceptibility test revealed that 34.7% of the isolates were MDR. Biofilm production was determined by Congo red assay (CRA) and Microtitre plate (MTP) method. CRA showed 89% as biofilm producers. MTP method showed 87% were biofilm-forming. MIC of Carbapenems was determined by the broth macrodilution method. It showed that 50% of isolates were resistant. (MIC) Minimum Inhibitory Concentration of 7 disinfectants against 10-selected MDR strong biofilm Pseudomonas isolates was determined using the broth microdilution method. It showed that the most effective disinfectants with the lowest MICs were the Sodium hypochlorite 5% and Povidone-Iodine 10%. The Real-time PCR was done on lasR and ndvb genes for the selected isolate (E20) before and after exposure to both effective disinfectants. The sample E20 showed a significant down regulation for lasR and ndvb genes with both effective disinfectants. Our study showed that Povidone-iodine 10 % at appropriate concentrations at less than 30 minutes has significant anti-biofilm activity against MDR Pseudomonas aeruginosa.