The mechanism and gene markers of head and neck squamous cell carcinoma (HNSCC), a common malignant tumor, have not yet been identified. The aim of this study was to identify the key genes and pathways associated with HNSCC and to further analyze its molecular mechanism and prognostic significance. In this study, the expression profile chip data GSE6631 from Gene Expression Omnibus (GEO) included paired HNSCC tumor and normal samples from 22 patients; the RNAseq tertiary dataset of HNSCC and corresponding clinical information from The Cancer Genome Atlas (TCGA) included biological information of 12 normal head and neck tissues and 111 HNSCC sample tissues. Differentially expressed genes (DEGs) were screened by R software, and the pathway enrichment analysis of DEGs was performed by DAVID, String, and Sytoscape software programs. Combining the GEO and the TCGA databases, we used bioinformatics technology to screen out 50 DEGs in HNSCC and enrich the biological functions and key pathways of HNSCC. Then we performed Gene Ontology (GO) enrichment analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, protein-protein interaction (PPI) analysis, and survival analysis on these DEGs. Using CMap, we identified candidate small molecules that might reverse HNSCC gene expression. Finally, four most important small molecules that could provide more reliable biomarkers for early diagnosis and individualized control of HNSCC were identified.