Chamiside A (1), a novel cytochalasan with a new 6/6/5-fused tricyclic core skeleton, was isolated from an endophytic fungus, Chaetomium nigricolor F5, harbored in the medicinal plant Mahonia fortunei. Its structure was unambiguously determined by extensive spectroscopic analyses, measurement of single-crystal X-ray diffraction, and electronic circular dichroism calculation. A biosynthetic pathway for the unique ring system in 1 was proposed. Compound 1 exhibited moderate antibacterial activity against Staphylococcus aureus.