Origanum vulgareleaves are collected from a local garden, Baghdad, Iraq. The hot and cold aqueous extract was prepared to perform an In vitro antibacterial and antibiofilm actions .Antibacterial activities on various antibiotic resistant bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Echerichia coli, Staphylococcus aurous and staphylococcus epidermidis) were tested by well diffusion method. The biofilm-forming ability of the tested microorganisms was determined by the Microtiter-plate method. Fresh hot extract showed the highest effect and the dried cold extract had the lowest effect against gram-negative and gram-positive bacteria . fresh hot extract decrease significantly the biofilm formation and showed good ability as an antibiofilm to all strains that tested..Antimicrobial activity of the extract seemed to be related to the aqueous extract preparation method and that appeared by the difference between fresh and dried leave extract activity against pathogens and it can be used as potential antibiofilm agent against the biofilm forming strains.