Phenolic metabolite profiling and identification using high performance liquid chromatography (HPLC) coupled to high resolution accurate mass spectrometry (HR-ESI-MS) with detection of negative ions was used for assaying the complex mixture of phenolics of an aqueous ethanol leaf extract of Eugeniajambos L. (Myrtaceae). Eight known polyphenolics were tentatively identified, and, in addition, three hitherto unknown flavonol-O-glycosides were detected in the extract. These unknowns were taken as the targets and isolated by means of consecutive polyamide S6, MCI gel and repeated Sephadex LH-20 column fractionation. The isolation and purification were monitored by HPLC/ESI-MS. The isolates were subsequently identified as quercetin 3-O-xylosyl-(1"' --> 2")-O-xyloside, myricetin 7-methylether 3-O-xylosyl-(1"' --> 2")-rhamnoside and myricetin 3',5'-dimethyl ether 3-O-xylosyl-(1"'-->* 2")-O-rhamnoside. All known metabolites were also separated by applying the same chromatographic techniques. ESI-MS, ¹H and ¹³C NMR spectra were then recorded, completely interpreted and confirmed by HR-ESI-MS and 2D NMR spectroscopy. In order to get information about biological activities of E. jambos the extract was tested for radical scavenging activity by DPPH and ORAC assay. In addition, its cytotoxicity was assessed by the neutral red assay against non-tumorigenic HaCaT keratinocytes and the human bladder carcinoma cell line 5637.