The present study was designed to investigate the effect of antimicrobial ethanolic extract of propolis (EEP) on gram negative and gram positive bacterial. The tested bacterial strains were Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Proteus as Gram negative, while Staphylococcus aureus and Streptococcus pyogenes consider as Gram positive. Four different types of propolis (Saudi, Turkish, Chinese and Egyptian) were used in this study. There was highly significant effect of Saudi, Egyptian and Turkish propolis on tested bacteria. The highest inhibition zone in Egyptian propolis was 12 mm with Proteus mirabilis at 10% concentration while the lowest inhibition zone was 1.6 mm with E.coli at 1.25% concentration at the same propolis. Proteus mirabilis showed the highest inhibition diameter which record 14 mm at 10% concentration in Saudi propolis while the lowest diameter was recorded as 2.6 mm at 1.25% concentration with P.aeruginosa. In Chinese propolis the Proteus showed the highest inhibition zone at 1.25 %, 2.50%, 5%, 7.50% and 10% concentration that recorded (5mm), (7.3mm), (9.6mm), (11mm) and (13 mm) respectively. In Turkish propolis the 10.5 mm was recorded as the highest inhibition zone at 7.5% and 10% concentration in Klebsiella pneumoniae, on the other hand, the lowest inhibition zone in S.aureus was 1.6 mm at 1.25% concentration. the antibacterial activity of propolis was concentration depends and depends upon its botanical origin