Using natural preservatives such as plant essential oils is a healthy and environmentally friendly approach that is a good alternative to chemical additives and helps maintain consumer health. Eucalyptus or the water gum plant with the scientific name Eucalyptus globulus belongs to the Myrtaceae family. This investigation was conducted to identify chemical compounds and evaluate the antibacterial activity of eucalyptus essential oil  (EEO) on a number of pathogenic microorganisms in vitro. In this study, the chemical compounds of eucalyptus essential oil were identified using a GC-MS device. Then, the effect of different concentrations of EEO (0, 2, 4, 6 μL/L) on some pathogenic bacteria and the microbial characteristics of the doogh samples were investigated during a 45-day storage period. The results of GC-MS analysis showed that 1,8-cineole (48.6%) and alpha-pinene (23.86%) constituted the highest percentage of EEO compounds. By adding 6 μl/l of EEO to the dough, no mold and yeast growth was observed throughout the storage period, and the sample containing 4 μl/l of EEO also contained an acceptable number of mold and yeast at the end of the storage period. EEO also reduced the total count of microorganisms. According to the results of this study, gram-negative bacteria were more resistant to the plant essential oil than gram-positive bacteria, and Salmonella typhi was identified as the most resistant and Bacillus cereus as the most sensitive bacteria in the disk method. The results of this study showed that EEO has appropriate antimicrobial activity against pathogenic strains. Therefore, by using this essential oil in the production of dough, in addition to increasing the shelf life of the product, it is possible to produce dough with suitable medicinal properties. However, in order to safely use EEO in the formulation of food products such as dough, further studies are necessary.