Pseudomonas aeruginosa is one of the most frequently isolated organisms from infected burn wounds and a significant cause of nosocomial infection and septic mortality among burn patients. In this animal study, three antiseptic agents which were Octenidine dihydrochloride (Octenisept®, Schülke & Mayr, Norderstedt, Germany), polyhexanide (Prontosan®, B. Braun, Melsungen AG, Germany) and povidon iodine (Betadine, Purdue Pharma L.P, Stamford, USA) were compared to assess the antiseptic effect of their applications on experimental burn wounds in in rats contaiminated with P. aeruginosa. All treatment modalities were effective against P. aeruginosa because there were significant differences between treatment groups and control groups. The mean eschar concentrations were not different between polyhexanide and povidon iodine groups, but there were significant differences between the octenidine dihydrochloride group and the other treatment groups, indicating that the Octenidine dihydrochloride significantly eliminated P. aeruginosa more effectively in the tissues compared to the to other agents. All treatment modalities were sufficient to prevent the P. aeruginosa invasion into the muscle and to cause systemic infection. In conclusion, Octenidine dihydrochloride is the most effective antiseptic agent in the treatment of the P. aeruginosa-contaminated burn wounds; Octenidine dihydrochloride can be considered as a treatment choice because of its peculiar ability of limit the frequency of replacing wound dressings.