Objectives: This study examined the impact of various abiotic factors, specifically temperature and precipitation, on the yield and composition (volatile profile) of essential oils (EO) from four Eucalyptus species, including Eucalyptus salmonophloia, Eucalyptus torquata, Eucalyptus lesouefii and Eucalyptus astringens. Additionally, the antimicrobial properties of these EO were assessed. Methods: The species were collected from five arboreta in Tunisia belonging to two climatic conditions (arid and semi-arid). EOs were extracted from the leaves using the hydrodistillation technique and analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The antimicrobial activity was evaluated by measuring the diameters of inhibition zones using the agar well diffusion method and by determining the minimum inhibitory concentrations (MICs). Results: The yield of Eucalyptus EOs varied from 0.12% to 4.63% (w/w, dry weight) depending on the species and the plant's growing location. 1,8-cineole (29.71% to 67.16%) was by far the major compounds in EOs of E. salmonophloia, E. lesouefii and E. astringens, however E. torquata was torquatone chemotype (33.41% to 44.78%). In general, the aridity increased the extraction yield of EO. Higher temperature and lower rainfall conditions enhanced the production of key compounds such as, 1,8-cineole and alpha-pinene, however, it decreases others compounds like spathulenol and viridiflorol. A notable antimicrobial activity was observed against all microbial strains tested, demonstrating both microbicidal and microbiostatic effects, particularly against Escherichia coli, Serratia marcescens, and Candida tropicalis. The EOs derived from the studied Eucalyptus species represent a valuable source of bioactive compounds, including 1,8-cineole, alpha-pinene, spathulenol, and beta-eudesmol. These compounds contribute to the oils' significant antimicrobial efficacy, offering the additional advantage of being a natural product. Conclusion: Our findings reinforce the notion that environmental factors may serve as a limiting factor in the production and availability of Eucalyptus EO for medicinal and industrial applications.