Two highly conjugated alkaloids xylaridines A (1) and B (2) were obtained as racemates from the fungus Xylaria longipes. They were resoluted into optically pure enantiomers, respectively. Their structures were determined by extensive spectroscopic analyses, X-ray diffraction, and equivalent circulating density (ECD) calculations. Compound 1 possesses a 5/6/6/5/5 fused ring system with unique 2-azaspiro[4.4]nonane substructure. The hypothesis of biosynthesis pathways for 1 and 2 was proposed.