Comprehensive Summary Talachalasins A—C (1—3), three new cytochalasans (CYTs), were isolated from deep‐sea‐derived fungus Talaromyces muroii sp. SCSIO 40439. The structures of 1—3, including absolute configurations, were determined based on HRESIMS, NMR spectroscopic data, ECD comparison, and single crystal X‐ray diffraction analysis. Talachalasins A—C (1—3) represented the first examples of cytochalasans possessing a unique 16β‐methyl group, distinct from >500 reported CYTs. Talachalasin C (3) was an unprecedented 20,21‐seco‐cytochalasans characterized by a 2‐oxabicyclo[3.3.1]nonan‐3‐one unit. Talachalasin A (1) displayed moderate cytotoxic activity against four tumor cell lines (IC50 3.40—10.02 μM). Talachalasin B (2) showed comparable anti‐RSV and HSV‐1 activities (IC50 12.5 μM or IC50 20.0 μM).