Two new chlorinated preussomerins, chloropreussomerins A and B (1 and 2), together with nine known preussomerin analogues, 3-11, were obtained from the endophytic fungus Lasiodiplodia theobromae ZJ-HQ1. Their structures were elucidated by a combination of spectroscopic analyses. The absolute configurations of 1 and 2 were both determined by single-crystal X-ray diffraction using Cu Kα radiation. Chloropreussomerins A and B (1 and 2) are the first chlorinated compounds in the preussomerin family, and preussomerin M (3) is reported for the first time as a natural product. Compounds 1 and 2 showed potent in vitro cytotoxicity against A549 and MCF-7 human cancer cell lines, with IC50 values ranging from 5.9 to 8.9 μM, and compounds 4-7 exhibited significant bioactivity against A549, HepG2, and MCF-7 human cancer cell lines, with IC50 values of 2.5-9.4 μM. In the antibacterial assay, compounds 1, 2, 5-7, and 11 exhibited significant activities against Staphylococcus aureus, with MIC values between 1.6 and 13 μg/mL.