The aims of our research include the phytochemical characterization of the essential oils extracted from different lavender species (Lavandula vera L., L. intermedia L., L. pyrenaica DC., and L. stoechas subsp. stoechas) that are important from therapeutic and economic aspects, as well as the optimization of the tube dilution method for microbiological investigation of the effect of essential oils on the selected Pseudomonas strain. The chemical and percentage compositions of the essential oils were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The main component of the essential oils in the majority of lavender species was linalool, while L-fencone was identified in the largest amount in L. stoechas subsp. stoechas. Tube dilution is one of the most frequently used methods for microbiological investigation. Its advantages are that it is easy to perform and provides information about minimum bactericidal concentration (MBC). Because of the lipophilic character of essential oils, the tube dilution method should be optimized for the detection of antibacterial activity of these plant extracts, and, therefore, emulsions containing 0.2% polysorbate 80 were prepared from the samples. In each of the eight investigated essential oils an inhibitory effect was detected, and MBC values were in the range 12.5-50 microL/mL in seven cases.