Background <p>Petit grain (PGO), a citrus essential oil (EO), isolated from the twigs and leaves of bitter orange (<i>citrus arantiun</i>), has been shown to have different beneficial properties, such as antibacterial, anti-fungal, anti-viral, anti-inflammatory and anti-cancer properties. Hypopharyngeal carcinoma is typically treated with combination radiotherapy and chemotherapy (cisplatin). However, resistance to cisplatin may lead to other new treatments, such as immunotherapy, which can be well-tolerated and potentially beneficial.</p> Purpose <p>In this study, PGO was proposed as a natural growth-regulating molecule of the FaDu cell line from hypopharyngeal squamous cell carcinoma.</p> Methods <p>The study used MTT assay, growth curves, Western blot, and FACS analysis to assess the impact of PGO on petit grain cell growth, protein levels, and cell cycle phases.</p> Results <p>The potent cytotoxic effect of PGO against FaDu was demonstrated by its low IC50. Daily administration of 0.025% (v/v) and 0.5% (v/v) PGO on FaDu cells resulted in a time and dose-dependent decrease in cell viability and growth. PGO caused a cell cycle arrest in the G1/S transition phase, which was verified by increased expression of cell cycle regulating proteins p27, p21 and p18, and a decrease of CDK2 and CDK4 proteins. PGO treatment also reduced the phosphorylation of the proliferative signals pAKT. Last but not least, PGO dramatically increased the sensitivity of FaDu cells to chemotherapy when combined with cis-platin, the usual treatment for squamous cell carcinoma.</p> Conclusion <p>Since PGO inhibits the growth of FaDu squamous carcinoma cells through a variety of combined mechanisms, it could be considered as a promising adjuvant therapy for this complex disease.</p> Graphical Abstract <p></p>