This research aims at separation of polyphenols from Jordanian olive oil mill wastewater which have possible applications in pharmaceutical industry. The phenolic compounds were isolated by silica column chromatography based on using different solvents after extracting the acidified solution with n-hexane and ethyl acetate. The structural elucidation of the separated compounds was achieved using 1H-NMR, 13C-NMR and mass spectrometry. The concentrations of these compounds were determined by GC–MS after derivatization with N,O-bis(trimethylsilyl) trifluoroacetamide. The concentrations of the main phenolic compounds in the olive mill wastewater which we could isolate were: ferulic acid (93.6 mg/L), trans-cinnamic acid (105.3 mg/L), p-coumaric acid (117.0 mg/L), vanillic acid (128.7 mg/L), caffeic acid (140.4 mg/L), tyrosol (210.6 mg/L) and hydroxytyrosol (315.9 mg/L).