Heat shock factor 1 (HSF1) is a transcription factor that regulates expression of heat shock protein (HSP) genes in response to stress. HSPs are expressed at high levels in a wide range of tumors. It has been reported that HSF1 and HSPs are associated closely in tumorigenesis. In the present study, a screen was performed using a luciferase reporter under the control of a heat shock element to find inhibitors of HSF1 activity, and 2,4-bis(4-hydroxybenzyl) phenol (1), isolated from the rhizomes of Gastrodia elata, was identified as an active compound. This substance effectively inhibited HSF1 activity and decreased levels of HSP27 and HSP70. Compound 1 induced the degradation of HSF1 protein through dephosphorylation of HSF1 on S326, which decreases HSF1 protein stability. In addition, 1 also induced growth arrest and apoptosis of NCI-H460 human lung cancer cells. Markers of apoptosis, such as cleaved PARP and cleaved caspase-3, were detected after treatment with 1. Furthermore, cotreatment with 1 and conventional anticancer modalities such as paclitaxel, cisplatin, or ionizing radiation potentiated their effects on lung cancer cells. These results suggest that inhibition of HSF1 by 1 may help overcome resistance to conventional anticancer modalities in HSF1-overexpressed cancer cells.