Three new 12- or 13-membered-ring macrocyclic alkaloids, named ascomylactams A-C (1-3), along with the analogues phomapyrrolidone C (4) and phomapyrrolidone A (5) were isolated from the mangrove endophytic fungus Didymella sp. CYSK-4. Their structures were elucidated by analysis of extensive spectroscopic data and mass spectrometric data. The structures and absolute configurations of 1 and 2 were determined by single-crystal X-ray diffraction experiments, which represents the first crystal structures described for a (6/5/6/5) tetracyclic skeleton fused with a 12- or 13-membered-ring macrocyclic moiety. The configurations of phomapyrrolidone C (4) and phomapyrrolidone A (5) were revised by detailed analysis of the NMR data. In a cytotoxic assay, compounds 1 and 3 showed moderate cytotoxicity against MDA-MB-435, MDA-MB-231, SNB19, HCT116, NCI-H460, and PC-3 human cancer cell lines, with IC50 values in the range of 4.2-7.8 μM. © 2019 American Chemical Society and American Society of Pharmacognosy.