The aim of this study was to investigate the chemical components and evaluate the antifungal activity of Origanum acutidens essential oil. The aerial parts of O. acutidens were collected, and the hydrodistillation method was used to extract the essential oil. Gas chromatography-mass spectrometry (GC-MS) analysis was performed to determine the chemical composition of the essential oil. The main components identified were α-terpineol (4.76%), p-cymene (7.6%), linalool (14.82%), and carvacrol (49.4%). The essential oils were tested against two pathogens in vitro experiments to evaluate their antifungal activity. Different concentrations of the essential oil were applied, and the inhibition of mycelial growth was measured. The results demonstrated that the essential oil exhibited antifungal properties against both pathogens. At a dose of 4.8 µL/Petri dish, the mycelial growth of both pathogens was completely inhibited. However, Sclerotinia sclerotiorum showed higher tolerance to the essential oil compared to Phytophthora infestans. Furthermore, a dose-effect study was conducted as a part of this research. The LC50 values (lethal concentration at which 50% of the pathogens' growth is inhibited) for P. infestans and S. sclerotiorum were calculated as 0.982 µL/Petri and 1.61 µL/Petri, respectively. The study concluded that the essential oil of O. acutidens has the potential to be a natural antifungal agent, particularly against S. sclerotiorum and P. infestans. However, further research is needed to investigate the mechanisms of action and explore potential applications of this essential oil in managing plant diseases.