Fruits and vegetables are highly consumed because they are rich sources of essential vitamins, minerals, fiber, and antioxidants, which are vital for our health. However, they are often vulnerable to attacks by fungi, particularly of the Penicillium genus, leading to significant economic losses. In response, this study examines the antifungal properties of clove essential oil (Syzygium aromaticum) on two types of phytopathogenic fungi, namely Penicillium brevicompactum and Penicillium expansum, through both lab experiments and computer simulations (in vitro and in silico). The findings indicate that the essential oil inhibited the growth of the fungi, with minimum inhibitory concentrations of 0.312 µL/mL for P. brevicompactum and 0.156 µL/mL for P. expansum. Moreover, sublethal concentrations of the oil resulted in inhibition rates of 58.3% ± 2.8% for P. brevicompactum and 51.7% ± 3.2% for P. expansum. Additionally, the essential oil-induced changes in the fungi's structure increase the permeability of their cell membranes and reduce spore germination, particularly in P. brevicompactum. Lastly, the study employed computer simulations to explore the potential morphological targets affected by the essential oil's components. The results suggest that clove essential oil could be a promising alternative for controlling phytopathogenic fungi and mitigating post-harvest losses, such as using it in edible coatings, which is important in the food industry.