The medicinal efficacy and low adverse effects of herbal remedies make them popular. Due to the presence of bioactive substances as 1,8-cineole, flavonoids, terpenoids, tannins, and phenolic compounds, Eucalyptus globulus is a significant medicinal plant recognized for its pharmacological activity. Using both in vitro and in vivo experimental settings, the current work attempts to assess the dose-dependent antibacterial and anti-biofilm activities of Eucalyptus globulus essential oil. Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were among the bacteria against which antimicrobial activity was assessed using the agar well diffusion technique. The microtiter plate test was used to assess anti-biofilm activity. An experimental animal model was used to assess in vivo activity. To find the dose-dependent response, several essential oil concentrations (25 µl, 50 µl, 75 µl, and 100 µl) were employed. Significant antibacterial action was demonstrated by the results, with a larger zone of inhibition at higher doses. Additionally, essential oil significantly inhibited the production of biofilms. An in vivo investigation revealed a decrease in the microbial load and an improvement in the state of infection. The results show that Eucalyptus globulus essential oil has strong antibacterial and anti-biofilm action that is dose-dependent, making it a promising natural medicinal agent for the treatment of microbial illnesses.