This study is focused on the potential antimicrobial activity of honey phenolic compounds against Gram positive and Gram negative bacteria. For this purpose phenolic compounds were isolated from 33 Iranian honeys obtained from different botanical and geographical origins using solid-phase extraction (SPE). Characterization of honey extracts was carried out by HPLC-DAD-ESI-QTOF/MS. Minimum inhibitory concentration (MIC) and inhibition zone were used to determine the antimicrobial capacity of honey phenolic extracts against Gram positive (Staphylococcus aureus and Enterococcus faecalis) and Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The results shown that E. coli was the most sensitive bacteria and P. aeruginosa the most resistant strain. Moreover, five phenolic extracts shown lower MIC values than the whole honey pointing out that honey phenolic fraction may exert antimicrobial activity by itself and be a source of bioactive compounds to develop functional ingredients.