Propolis is a resinous natural hive product derived from plant exudates collected by honeybees. Due to biological and pharmacological activities, it has been extensively used in folk medicine. The present study was designed to investigate the chemical composition, sub-chronic toxicity, antimicrobial activity of Iranian propolis ethanolic extract, which has not been studied previously. One hundred and nine compounds were identified by gas chromatography-mass spectrometry analysis. Forty-five days subchronic toxicity of oral propolis extract was investigated in male rats. During the study no significant behavioral and clinical toxicity has been seen in animals however, hematologic, blood biochemistry and histopathologic data studies exhibited some significant differences between the groups. The ethanolic extract of propolis inhibited the growth of all examined microorganisms including bacteria and fungi with the highest antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis. (c) 2006 Elsevier Ltd. All rights reserved.