Under the guidance of HPLC-DAD analysis, ten new chromones featuring a rare [6,6]-spiroketal moiety, namely chaetovirexylariones A-J (1-10), together with two known congeners (11-12), were isolated from coculture of the endophytic fungi Chaetomium virescens and Xylaria grammica, from the rhizome of the medicinal plant Smilax glabra Roxb. Their structures were elucidated via a combination of NMR and HRESIMS data, and the absolute configurations of 1-10 were determined by the chemical conversion and single-crystal X-ray diffraction (Cu Kα) experiments, as well as the comparison of the experimental and calculated electronic circular dichroism (ECD) data. Compound 6 is the first report as a racemate of this type of natural product. Compound 10 represents the first example of a [6,6]-spiroketal chromone bearing a 5-amino-3-methyl-2-pentenoic acid fragment. Compound 8 demonstrated a reduction in PTX resistance of SW620/AD300 by a factor of 45, and had the potential to be an effective P-gp inhibitor and an antitumor chemotherapy sensitizer.