Background Propolis is a resinous product accumulated from several plant sources that possess a wide range of therapeutic properties, including anti-cancer activities. However, the role of honeybee-produced propolis on head and neck squamous carcinoma (HNSCC) is not well understood. The aim of this study was to investigate the effects of Apis mellifera propolis on apoptosis and invasiveness in HNSCC cell lines. Methods Ethyl acetate extract of propolis (EAEP) was prepared from A. mellifera beehives using liquid-liquid extraction. High-performance liquid chromatography coupled with electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) was used to determine the flavonoids in EAEP. Isogenic HNSCC cell lines derived from primary (HN30 and HN4) and metastatic site (HN31 and HN12) were used in this study. The cytotoxicity, apoptosis, invasion, and MMP activity of EAEP on HNSCC cells were determined using an MTT assay, flow cytometry, Matrigel invasion assay, and gelatinase zymography, respectively. Results We found that EAEP exhibited cytotoxic activity and induced apoptosis in the HNSCC cell lines. Furthermore, EAEP significantly decreased HNSCC cell invasion by reducing MMP-2 and MMP-9 activity. Two flavonoids, galangin and apigenin, were identified in EAEP by HPLC-ESI-TOF-MS. The results suggest that EAEP promotes apoptosis and exerts anti-invasion potential by inhibiting MMP-2 and MMP-9 activity in HNSCC cell lines. These inhibitory effects may be mediated by galangin and apigenin.