One new flavonoid named 5,7-dihydroxy-3,6,3`-trimethoxyflavone (1), together with eight known phenolic compounds; 3,4-dihydroxy methyl benzoate (2), 3,4-dihydroxybenzoic acid (3), isorhamnetin (4), quercetin (5), myricetin (6), isorhamnetin-4`-O -D-glucopyranoside (7), quercetin-4`-O -D-glucopyranoside (8) and quercetin-3,4`-O -D-diglucopyranoside (9) were isolated from the flowers of Cucurbita pepo L. for the first time except 3 which was previously isolated from the plant. Their structures have been established on the basis of physical, chemical and spectroscopic methods in addition to comparison with literature data and/or authentic samples. The methanolic and ethyl acetate extracts were evaluated for their antimicrobial activity.