A pair of new enantiomeric alkaloid dimers, (+)- and (-)-pesialoxazine A (1), with an unprecedented symmetric spiro[oxazinane-piperazinedione] skeleton, consisting of 22 carbons and 12 heteroatoms, were isolated from a Pestalotiopsis sp. fungus derived from a soft coral. Separation of the enantiomeric alkaloid dimers was achieved by chiral HPLC. Their structures including absolute configurations were elucidated on the basis of a comprehensive analysis of their spectroscopic and X-ray diffraction data and CD calculations. (+)-Pestaloxazine A exhibited potent antiviral activity against EV71 with an IC50 value of 14.2 +/- 1.3 mu M, which was stronger than that of the positive control ribavirin (IC50 = 256.1 +/- 15.1 mu M).