Volatile oil extracted from fermentation broth of <i>Ganoderma lingzhi</i> by hydrodistillation was analyzed based on gas chromatography-mass spectrometry (GC-MS). Its antitumor activity was tested on K562, SW620, A549, HepG2 cells<i> in vitro</i>. In addition, the antioxidant activity of the oil was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. In total, 16 constituents were identified accounting for about 99.99% of the total volatile oil in the fermentation broth of <i>G. lingzhi.</i> Among these components, 1-propanol (33.33%), phenylacetaldehyde (24.24%), 2-hexyl-1-decanol (12.12%) were found to be the major constituents. The antitumor results showed that the IC<sub>50</sub> of the inhibition to the proliferation of K562, SW620, A549, HepG2 cells were 32.2, 78.9, 96.4, 99.0 μg/mL, respectively. And the oil could inhibit the proliferation of K562 cells by apoptosis induction and cell cycle arrest at S phase. Moreover, the oil exhibited radical scavenging activity (IC<sub>50</sub> = 0.1469 mg/mL) in DPPH assay.