Background and aim: Pseudomonas aeruginosa is an opportunistic pathogen affecting immunocompromised patients resulting in multiple life threatening infections. The multidrug resistant (MDR) P. aeruginosa strains represent a major challenge to clinical therapy, however, no effective vaccine is currently available in the market. Here, we aimed to develop novel polyvalent irradiated P. aeruginosa vaccine to protect immuno-compromised patients. Materials and methods: We developed polyvalent irradiated P. aeruginosa vaccine using cobalt 60. Results: Inhibited pathogen viability but retained antigenic expression functionally. Mice were vaccinated by the developed vaccine by intranasal, intramuscular and subcutaneous route of administration followed by challenge test. The protective efficacy of the novel P. aeruginosa vaccine reached up to 95%. This significant protection was mainly associated with measurable antiserum opsonic killing activity. Conclusion: The novel vaccine provides a promising strategy of both prophylactic and therapeutic approaches for immunocompromised patients against MDR P. aeruginosa.