Two novel compounds including a cyclohelminthol type polyketide (namely oxaleimide K,1) and a maleimide derivative (namely peniroquefortine A,2), and a new natural product (namely 2-(acetylamino)-N-[(1E)-2-phenylethenyl]-acetamide,3), together with four known compounds (4–7), were isolated and identified from fungusPenicillium roqueforti, which was separated from the root soil ofHypericum beaniiN. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures including absolute configurations were mainly established by the NMR spectroscopy analyses and single-crystal X-ray diffraction experiment. Compound1represents the second example of a cyclohelminthol type polyketide, which features a rare 6/6/5/5 tetracyclic system and a branched aliphatic chain containing a terminal olefin (oct-1-en-3-yl) moiety, and compound2possesses an unprecedented carbon skeleton that is uniquely defined by a maleimide moiety linked to the respective 4-methylene-2-(3-methylbut-2-en-1-yl)-phenol andpara-substituted aromatic moieties via the carbon-carbon bonds. Remarkably, the absolute configuration of a cyclohelminthol type polyketide as exemplified by compound1is determined by the single-crystal diffraction analysis for the first time, highlighting anE-configuration for the linkage of a succinimide moiety and a tetrahydrofuran moiety for1rather than aZ-configuration as previously reported in the biosynthesis study, which gives a new insight into the structural elucidation of this category of polyketides. Additionally, compound1exhibited significant cytotoxic activity against multiple tumor cells, especially against the Farage and SU-DHL-2 cells (IC50< 20 µM, 48 h). Further mechanism study revealed that compound1significantly induced cell cycle arrest in Farage and SU-DHL-2 cells by causing abnormal ROS level and triggering oxidative stress.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s13659-023-00381-4.