Essential oils are mixtures of volatile, low-viscosity compounds, most of which are terpenes (monoterpenes and sesquiterpenes). Good solubility of essential oils in other vegetable oils and easy absorption through the skin allows them to be used in medical massage and aromatherapy. The most common compounds in the essential oil of prickly juniper (Juniperus oxycedrus), phoenician juniper (Juniperus phoenicea), laurel (Laurus nobilis), rosemary (Rosmarinus officinalis) and mastic tree (Pistacia lentiscus) are monoterpenes, while in the essential oil of sage (Salvia officinalis) are sesquiterpenes. Using hydrodistillation, essential oils of these plants were obtained and characterized by various methods of analysis such as thin layer chromatography, gas chromatography-mass spectroscopy, Fourier-transformed infrared spectroscopy and microbiological analysis. Chemical composition and antimicrobial activity of tested essential oils were analytically determined. The results obtained by different analytical methods are comparable and they describe chemical composition of essential oils. The best known method of microbiological analysis is the disk diffusion test which examined the antimicrobial activity of essential oils. Depending on the chemical composition of essential oil, in disk diffusion test the effect on bacteria Escherichia coli and Bacillus subtilis and yeast Candida lipolytica is different. The results showed that the essential oil of Mediterranean laurel has the best antimicrobial activity for tested bacteria, and the essential oil of mastic tree for tested yeast. The obtained results are comparable with the literature with small deviations in the chemical composition and representation of the components of essential oils.