Two new 12- or 13- membered-ring macrocyclic alkaloids ascomylactam D and E (1 & 2), and a pair of new enantiomer (+)- and (−)- didymetone (3) were purified from the mangrove endophytic fungus Didymella sp. CYSK-4. Their structures and absolute configurations were determined by extensive spectroscopic analysis, single-crystal X-ray diffraction, ECD and 13C NMR calculations. Compound 2 exhibited significant cytotoxicity against human A549 and KYSE 150 cancer cell lines with IC50 values of 2.8 μM and 5.9 μM, respectively. © 2023