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Introduction: Pseudomonas aeruginosa is a key opportunistic pathogen causing severe acute and chronic nosocomial infections in immunocompromised or catheterized patients. It is prevalent in burn wound infections and is generally multi-drug resistant.This study evaluated antimicrobial activity of ZnO nanoparticles, catechin and EDTA on the most common pathogen bacteria, Pseudomonas aeroginusa, by standard microdilution test depending on NCCLS. Material and Methods: Zinc acetate dehydrate and ethilen glycol (EG) were dissolved in 50 ml distilled water with a ratio of 10/1 (Zn/EG). The solution was heated at 80 0 C until a little gel of zinc acetate was obtained. Then, the gel was dried in the oven for 12h. The dried gel was calcinated at 300 0 C for 24h. The size and type of these nanoparticles were characterized by scanning electron microscopy (SEM) and X-Ray-: Diffraction (XRD). Pseudomonas aeroginusa strain (ATCC 27853) were cultured on nutrient agar medium (NA) for 24h at 37 0 C. The microbial suspension (1×10 6 cells/ml) was prepared. Minimum Inhibitory Concentration (MIC) test for ZnO, EDTA and Catechin were evaluated by microdilution test. Results: Evaluation of morphology and measurement of the size of the ZnO showed that nanoparticles were spherical with a diameter of 30-90 nm. MIC of ZnO, EDTA and Catechin on pseudomonas aeroginusa was 0.0003, 24.92 and 7.24 μg/ml, respectively. Conclusion: In this study, ZnO nanoparticles were synthesized using the chemical method. It was revealed that it has an optimal antimicrobial activity in low concentration as compared with Catechin and EDTA. It is recommended that catechin and ZnO nanoparticles are good candidates for eliminating some contaminations including Pseudomonas aeroginusa in medical industrial.