The objective of the present study was to identify chemical constituents of volatile oil from Pyrolae herba (PHVO) and evaluate the antiproliferative activity of PHVO against SW1353 human chondrosarcoma cells. The volatile oil from Pyrolae herba was prepared by hydrodistillation and characterized by gas chromatography-mass spectroscopy (GC-MS). A total of 12 components in PHVO were identified representing 81.62% of the total integrated chromatographic peaks. The major compounds were found to be n-hexadecanoic acid (29.29%), cedrol (17.08%), 6,10,14-trimethyl-2-pentadecanone (9.59%) and cis-9-octadecadienoic acid (8.23%). The antiproliferative activity of PHVO against SW1353 cells was investigated using MTT assay, flow cytometry and western blot analysis. Our results demonstrated that PHVO inhibited SW1353 cell viability in a dose- and time-dependent manner. Furthermore, PHVO treatment decreased the number of cells entering the S phase and caused a reduction in the expression of cyclin D1, cyclin-dependent kinase (CDK)4 and CDK6, whereas it caused an increase in the expression of p21. PHVO demonstrated potent antitumor activity against SW1353 cells, suggesting its potential use as a therapeutic agent in the treatment of chondrosarcoma.