The fermentation product of Cordyceps sinensis mycelium Cs-4 was commonly used as alternative substitutes of natural C. sinensis. Massoia lactone is the dominant component in the volatile oil of Cs-4 mycelium. In this research, we present a high performance liquid chromatography (HPLC) method for the quantitation of massoia lactone in Cs-4 mycelium. The high and stable contents of massoia lactone with values of 2.98-3.77mg/g, indicated that massoia lactone could be considered as a marker for the quality assessment of this product. The results of MTT and CCK-8 assay showed that Cs-4 mycelium volatiles exhibited cytotoxicity against eight malignant tumor cells (IC50 =6.0-49.8μg/ml) in comparison to the anticancer drug 5-fluorouracil (IC50 =17.0-425.3μg/ml), and massoia lactone might be the chemical basis for the anticancer effects of Cs-4 mycelium. Compared to the commercial drugs paclitaxel and docetaxel (IC50 =253-1973μg/ml), the Cs-4 mycelium volatiles and massoia lactone were discovered to possess inhibitory to taxol-resistant cell lines (IC50 =1.5-8.6μg/ml). PRACTICAL APPLICATIONS: Considering that there is still a lack of marker components distinctive to Cs-4 mycelium, the HPLC method represents a useful tool for the quality evaluation of Cs-4 mycelium. Moreover, the volatile oil of Cs-4 mycelium and massoia lactone have prominent anticancer property in vitro. It gives a clue that Cs-4 mycelium, the volatile oil and massoia lactone could be potentially employed in the food and medical industries for its anticancer applications.