Abstract Purpose: The EORTC-90111-24111 phase II window study evaluated afatinib versus no preoperative treatment in patients with primary squamous cell carcinoma of the head and neck (HNSCC). We investigated afatinib-induced tumor and microenvironment modifications by comparing pre- and post-treatment tumor biopsies. Patients and methods: Thirty treatment-naïve patients with primary HNSCC were randomized. Twenty-five patients received afatinib for 14 days before surgery (40mg 1x/day) and five patients were attributed to the control arm. Biopsies were taken at work-up and during surgery. Good quality RNA samples were used for omics analyses. The control arm was enlarged by samples coming from our previous similar window study (Schmitz et al. 2013). Results: Immunohistochemistry analyses of afatinib-treated tumor biopsies showed a decrease in pEGFR (P≤.05) and pERK (P≤.05); an increase in CD3+ (P≤.01) and CD8+ (P≤.01) T-cell infiltration, and in CD3+ (P≤.05) T-cell density. RNA sequencing analyses of afatinib-treated tumor samples showed upregulation of inflammatory genes and increased expression scores of signatures predictive of response to PD-1 blockade (P≤.05). In post-treatment biopsies of afatinib-treated patients, two clusters were observed. Cluster 1 showed a higher expression of markers and gene sets implicated in epithelial-to-mesenchymal transition (EMT) and activation of cancer-associated fibroblasts (CAFs) compared to cluster 2 and controls. Conclusion: Short-term treatment with afatinib in primary HNSCC induces CD3+ and CD8+ tumor infiltration and, in some patients, EMT and CAFs activation. These results open perspectives to overcome resistance mechanisms to anti-HER therapy and to potentiate the activity of immune checkpoint inhibitors.