Mangrove-associated fungi are important sources for the discovery of new bioactive natural products. Three new isocoumarins (1–3) and one new pyrone derivative (4) were isolated from the ethyl acetate extract of the fermentation broth of the mangrove endophytic fungusPhomopsissp. DHS-11. Nuclear magnetic resonance (NMR) spectroscopy (one-dimensional and two-dimensional) and mass spectrometry were used to determine the structures of these new compounds. The absolute configurations for the new isocoumarins1–3were determined by comparing their experimental and calculated electronic circular dichroism (ECD) spectra, while the configuration for the new pyrone-derivative4was tentatively solved by comparison of its13C NMR data with reported data. In the biological activity test, compounds1and3showed cytotoxic activity against HeLa cells with IC50values of 11.49 ± 1.64 µM and 8.70 ± 0.94 µM, respectively. The initial structure and activity relationship (SAR) analysis revealed that the length of the side chain at C-3 for isocoumarin-type compounds1–3could affect the cytotoxicity against HeLa cells. Compound4exhibited cytotoxic activities against human hepatoma cells HepG2 with an IC50value of 34.10 ± 2.92 µM. All compounds have no immunosuppressive activity.