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 (1H–1H 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 g/ml respectively.