Niclosamide is an oral chlorinated salicylanilide antihelminthic agent with potential anticancer activity suggested in several cancer types, however, its anticancer action and likely molecular mechanism in malignant oral cells remain unclear. In the present study, we demonstrated that ALDH+ human oral squamous cell carcinoma (OSCC) cells are characterized by upregulated expression of the pluripotency transcription factors OCT4, Nanog and Sox2, as well as exhibit enhanced cancer stemness, as demonstrated by enhanced tumorsphere formation. We also showed that niclosamide effectively inhibits activation of the Wnt/β-catenin signaling pathway by targeting multiple components of this pathway, including downregulating the expression β-catenin, Dishevelled 2 (DVL2), phosphorylated glycogen synthase kinase-3β (p-GSK3β) and Cyclin D1, in human OSCC SCC4 and SCC25 cell lines, as well as reduced the formation of primary and secondary tumorspheres. In addition, we showed that niclosamide inhibits the epithelial-to-mesenchymal transition (EMT), migration and colony formation of the OSCC cells, by dose-dependently upregulating E-cadherin and the tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels, while reducing the expression levels of vimentin, snail, MMP2 and MMP9 mRNA. These anticancer activities of niclosamide were similar to those caused by interference with nuclear β-catenin/c-Myc expression using the siRNA transfection. Finally, we demonstrated that niclosamide inhibits cisplatin-induced OSCC stem cell enrichment and enhances sensitivity to cisplatin in ALDH+ tumorspheres. These experimental data, combined with accumulated evidence, are suggestive of the potential and efficacy of niclosamide in the treatment of OSCC.