Aims: Today, bacterial biofilms contribute to 65% of nosocomial infections worldwide. One of the most common pathogens that can form biofilm is Pseudomonas aeroginusa. Therefore, the present study was aimed to evaluate the antimicrobial effects of catechin and ethylenediaminetetraacetic acid (EDTA) on planktonic and biofilm cells of P.aeruginosa standard strain. Methodology and results: Standard strains of P. aeruginosa (ATCC 27853) were cultivated in nutrient agar medium for 24 h at 37 °C. The MICs values of EDTA, catechin and imipenem antibiotics on P. aeruginosa were determined with micro dilution test. Then, the biofilm of this bacterium was grown and finally the influences of these agents on biofilm inhibition were evaluated by colorimetric MTT and ATPase release assays. One way analyses of variance and then Fisher's least significant difference test were carried out to compare the different groups. The MIC values of catechin and EDTA on P. aeruginosa were 7.24 and 24.92 (μg/mL), respectively. Colorimetric assay with MTT showed that EDTA, and catechin inhibited biofilm formation significantly. ATPase assay indicated that the amount of released ATP from EDTA and catechin groups were significantly lower than the control group. Also, there was a significant difference between the EDTA and catechin groups with respect to the amount of the released ATP. Conclusion, significance and impact of study: Our findings showed that EDTA and catechin can inhibit the growth of planktonic and biofilm cells of P. aeruginosa. From the results of the present study, we suggest using these agents to reduce or inhibit bacterial contamination of medical devices.