Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD−L1 expression/PD−1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.