Two new acetylated flavonol glycosides, quercetin 3-O-[(2,4-diacetyl--L-rhamnopyranosyl)-(16)]-2,4-diacetyl--D-galactopyranoside (1) and quercetin 3-O-[(2,4-diacetyl--L-rhamnopyranosyl)-(16)]-3,4-diacetyl--D-galacto-pyranoside (2), in addition to two known acetylated quercetin glycosides quercetin 3-O-[(2,3,4-triacetyl--L-rhamnopyranosyl)-(16)--D-galactopyranoside (3) and quercetin 3-O-[(2,3,4-triacetyl--L-rhamnopyranosyl)-(16)-3-acetyl--D-galactopyranoside (4), were isolated from the aerial part of Centaurium spicatum (L.) Fritsch (Gentianaceae). Structure elucidation, especially the localization of the acetyl groups, and complete 1H- and 13C-NMR assignments of these biologically active compounds were carried out using one- and two-dimensional NMR measurements, including 1H- and 13C-NMR, DEPT-135, H–H COSY, HMQC and HMBC, in addition to HR-FAB/MS experiments.