This study aims to evaluate the expression profile and clinical value of the S100 family in head and neck squamous cell carcinoma (HNSCC). The expression patterns, clinicopathological features, prognostic significance and underlying correlations of S100 family genes in HNSCC were determined by bioinformatics analysis with the application of several databases, including the The Cancer Genome Atlas (TCGA) and Oncomine for differential expression gene (DEG) analysis, and a series of analysis tools, including Database for Annotation, Visualization and Integrated Discovery (DAVID), cBioPortal, Kaplan-Meier Plotter, Tumor Immune Estimation Resource (TIMER) and R software packages. The results from the study demonstrated that S100A4, S100A10, and S100A13 may act as prognostic markers through overall survival (OS), disease-free survival (DFS) and tumor infiltrating immune cell enrichment and a prognostic S100 family gene model comprising S100A1-A4, S100A8, S100A10, S100A12, and S100A13 was identified. The messenger RNA (mRNA) expression of S100A1, S100A9, S100A14, and S100A7A was significantly different in HNSCC patients, together with a high mutation rate of the S100 family was found. Evaluation of clinicopathological value demonstrated the heterogeneity of S100 family functions. S100A1, S100A7, S100A8, S100A9, S100A13, S100A14, and S100A16 were observed to significantly correlate with multiple biological processes (BPs) of HNSCC, including initiation, lymph node metastasis, and lymphovascular invasion. In addition, the S100 family were significantly associated with epithelial-mesenchymal transition (EMT)-related genes. This present study demonstrated that S100 family members are implicated in the initiation, progression, metastasis and survival of HNSCC.