In modern plant production, the protection of fruits in the field and after harvest from fungal diseases has always been a stumbling block for many producers. The mentioned problem is the main topic of research in the field of Phytomedicine, and the obtained results are trying to minimize the problem. Reducing the use of fungicides with the same mechanism of action and increasing awareness of the harmful effects of agrochemicals reduces the resistance of phytopathogenic fungi to preparations which are available, but limited in number. Since fungicides are becoming increasingly ineffective, they are withdrawn from the market, which is why the attention of modern research has been focused on finding environmentally friendly compounds with a wide spectrum of action in the lowest concentrations and a mechanism of action different from available fungicides. Such an environmentally friendly alternative to the biological control of phytopathogens is increasingly becoming an indispensable part of Phytopharmacy. It is known that plant production is hampered by damage caused by resistant phytopathogens, and among them the phytopathogenic fungus Alternaria alternata stands out, known as an economically important pathogen that, in addition to crops, also causes great damage to stored fruits. This fungus causes early blight of tomato fruits in field, and the disease was named by characteristic symptoms. As the disease progresses in tomato crops, defoliation of the leaves occurs, and the entry of infected fruits into warehouses leads to the decay of the fruits due to rot, which is why the control of this fungus is of great importance. Also, this widespread pathogen is mycotoxigenic, and often a source of allergens, which can have a harmful effect on human health. In order to reduce the pressure of economically important pathogens such as A. alternata, biological control with essential oils is one of the innovative options. Essential oils are intensively researched and in recent years, in many studies, they have been shown to be a potential alternative to the fungicides used so far. Since they are known to have antifungal properties, the research of this paper was conducted with the same aim, in which the antifungal effect of three essential oils of tea tree, rosemary and thyme was tested in vitro on the phytopathogenic fungus A. alternata. Testing of the effect of the volatile phase of essential oils on the growth and development of the fungus was carried out using the disc diffusion method, and the effect of the oil was quantified by measuring the surface of the mycelium of the fungi and determining changes in the microstructural levels of the pathogen by stereo- and light microscopy. Seven-day exposure of the pthogen to the volatile phase of thyme essential oil significantly inhibited the surface of the mycelium by 91.3% compared to the fungi growth in the control, while rosemary and tea tree oils did not significantly inhibit the pathogenic fungus (16.7% and 0.7%). After conducting microscopic analysis, morphological changes of hyphae from the variant with essential oils were determined in comparison with hyphae from the control variant, especially in the variant with thyme essential oil, where a degenerative pattern of hyphal growth was determined compared to the control hyphae. Deformations of the apical parts of individual hyphae were determined, especially in the variant with rosemary oil, while tea tree oil showed the weakest effect on the pathogen's microstructures. It can be concluded that essential oils in the volatile phase show a fungistatic effect with an emphasis on the significant effect of thyme oil and the insignificant effect of rosemary and tea tree on the growth and development of the pathogen A. alternata. Given that the rapid development of resistance to conventional chemical control measures is a major problem in the control of the phytopathogenic fungus Alternaria alternata, the research results contribute to the development of eco-friendly methods and compounds in the alternative control of pathogens similar to the tested fungus. In vivo studies are needed to investigate the plant–pathogen–essential oil interaction for a more selective evaluation of the fungistatic effect, especially of thyme oil.