Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p = 0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p = 0.0010) with MMP-9 expression. The CD44+ invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.