Biofilm formation in pathogenic bacteria defends them from antibiotics and the host's immune system. In the course of our search for new inhibitors of biofilm formation in Mycobacterium species, we isolated the sesterterpenes ophiobolin K (1), 6-epi-ophiobolin K (2), and 6-epi-ophiobolin G (3) from a culture of marine-derived fungus of Emericella variecolor. Ophiobolins 1-3 inhibited biofilm formation of Mycobacterium smegmatis with MICs of 4.1-65 μM, whereas these compounds did not show antimicrobial activity at the concentrations that showed anti-biofilm formation activity. Ophiobolin K (1) was also effective against the biofilm formation of M. bovis BCG and was able to restore the antimicrobial activity of isoniazid against M. smegmatis by inhibiting biofilm formation.