Fifty-three fungal strains belonging to 15 mainly Fusarium species were isolated from branched broomrape plants. Their virulence was assessed using a plastic bag system, and they were grown both in liquid and on solid media, extracted, and the extracts were chemically analyzed and biologically assayed to find new metabolites that inhibit germination of branched broomrape to estimate the production of fusaric and dehydrofusaric acids by Fusarium strains and evaluate their possible involvement as virulence factors and their practical use as biomarkers to make the selection of potential mycoherbicides easier, and to ascertain whether toxins affected mammals. Nine strains proved to be highly virulent and 18 strains produced fusaric and dehydrofusaric acid at concentrations from 4 to 165, and from 9 to 204 mg L−1 respectively. Fifteen extracts from solid cultures caused high mortality when assayed on brine shrimps. Five extracts from liquid cultures caused total inhibition of seed germination. Some strains may be considered as sources of new biocontrol agents but virulence was not always positively correlated to the production of phytotoxic compounds.