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Three new pyrrolobenzoxazine sesquiterpenoids, talatrachyoxazines A - C (1 - 3 ), together with fourteen known compounds (4 - 17 ), were isolated from the fungus Talaromyces trachyspermus EU23. Their structures were identified by spectroscopic evidence and mass spectrometry. The absolute configurations of 1 - 3 were determined by NOESY data and comparison of their calculated and experimental electronic circular dichroism (ECD) spectra. Compound 1 showed cytotoxic activity against HelaS3, KB, HT-29, MCF-7, and HepG2 cell lines with IC