Propolis has been found to possess antibacterial activity and this has been attributed to specific chemicals in its composition, which depends on the region where it was collected. Our study evaluated the antimicrobial activity of propolis collected from Ardabil province located at northwest of Iran against S. aureus and P. aeroginosa. Twenty propolis (Apis mellifera) samples were obtained from the beehives situated in different regions of Ardabil province, located at the northwest of Iran. The disc diffusion method was employed to test the antibacterial activity of extracts of propolis (EEP, CEP and AEP). S. aureus (PTCC 1431) and P. aeruginosa (PTCC 1707) were used in this investigation to test antimicrobial activity of propolis. The extraction of propolis, regardless of how it is extracted, had the significantly higher inhibitory effect on the Gram-positive bacteria S. aureus compared to P. aeruginosa (p< 0.001). Both MIC and MBC of EEP, AEP, and CEP on S. aureus determined 0.164 mg/ml and there was no statistically significant difference. On the other hand, for P. aeruginosa, the amount of MBC and MIC for the EEP, AEP, and CEP determined as 0.022 mg/ml, 0.082 mg/ml, and 0.041 mg/ml, respectively. In conclude, in accordance with literature data the appropriate concentration of propolis might be effective on Gram-positive infectious bacteria but it was inactive against the Gram-negative bacteria. In vivo evaluations are required to find out concise antimicrobial mechanism of propolis and its appropriate dose. In addition, there is need for recognition of the antimicrobial active components in the propolis extracts.