The chloroformic fraction of the roots of Centaurium spicatum L. afforded one new xanthone named 1,5,8 trihydroxy-3,6,7-trimethoxyxanthone (1) together with six known xanthones (2–7), one of them isolated for the first time from a plant source (2). One secoiridoid glucoside (8) was also isolated. The structures of the isolated compounds were established based on 1D and 2D (1 H–1 H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested for their antimicrobial and antiprotozoal activities. Compound 6 displayed moderate antifungal activity against Candida krusei and Cryptococcus neoformans with IC50 values of 12.8 and 17.9 mg/ml respectively.