Aphidicolin, characterized by a highly fused 6/6/5/6 tetracyclic diterpenoid skeleton, had been explored as a potential anticancer drug in clinical trials. However, its development has been constrained by poor solubility. The discovery of new aphidicolin derivatives offers promising prospects for anticancer drug development. In the present study, 37 new aphidicolin derivatives, designated as aphidicolins B1-B37 (1-37), together with 34 known analogues (38-71), were discovered from the ethyl acetate (EtOAc) extract of the deep-sea-derived fungus Botryotinia fuckeliana. Their structures, including absolute configurations, were determined by extensive analyses of spectroscopic data, the phenylglycine methyl ester (PGME) method, modified Mosher's method, and comparison of experimental and calculated electronic circular dichroism (ECD) data. Notably, aphidicolin B12 (12) features a 6/6/5/6/6 pentacyclic framework with an unprecedented γ-lactone ring E, while compound 31 contains a novel 6/6/5/6/5/5 hexacyclic system bearing an unprecedented tetrahydrofuran ring formed by an ether bridge between C-3 and C-6. All isolated aphidicolins were evaluated for their cytotoxic effects against T24 human bladder cancer cells. Among them, 11 diterpenoids showed stronger inhibitory activity than aphidicolin (60), especially compound 32, which exhibited an IC50 value of 1.9 μM, significantly more potent than 60 (IC50 = 27.6 μM). The structure-bioactivity relationships were also discussed. Further mechanistic studies revealed that 32 inhibits T24 cells proliferation by inducing cell cycle arrest in the G0/G1 phase, suggesting its potential as a therapeutic agent for bladder cancer treatment.