Biofouling, the colonization of submerged surfaces by living organisms, is a widespread global phenomenon causing serious problems and huge economic losses every year. Marine microorganisms have proved to be an important source of antifouling metabolites. In our preliminary experiment, we found the EtOAc extract of a culture broth of the halotolerant fungal strain Penicillium sp. OUCMDZ-776 exhibited significant antifouling activity toward laboratory-reared Bugula neritina larvae. Bioassay-guided fractionation of the extract led to the isolation of a new alkaloid penispirolloid A (1) possessing a unique spiro imidazolidinyl skeleton. On the basis of extensive spectroscopic methods and quantum chemical CD calculation, the planar structure and partial absolute configuration of 1 were determined. Compound 1 showed significant antifouling activity toward Bugula neritina larvae with EC50 of 2.40 lg/ml. The plausible biogenesis of 1 was also proposed.