The antimicrobial properties of essential oil from Lavandula sp. raise hopes related to its use in phytotherapy. This study aimed to evaluate the antimicrobial activity of essential oils from cultivars of L. angustifolia (‘Hidcote Blue Strain’, ‘Hidcote Blue’) and L. × intermedia (‘Phenomenal’, ‘Grosso’) grown in central-eastern Poland, that is, at the border of the northern lavender cultivation range. The chemical composition of the essential oils was determined by GC/MS. Essential oil concentrations (20, 10, 5, 2.5, 1.25, 0.6, 0.3, 0.16, 0.08, and 0.04 mg/mL) were tested to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) towards ten strains of Gram-positive bacteria, five Gram-negative bacteria, and eight yeasts in vitro culture. Essential oils from the Lavendula cultivars showed antimicrobial activity against all microorganisms analysed. The yeasts were characterised by higher sensitivity to lavender oil compared to bacteria, while Gram-positive bacteria were more sensitive than Gram-negative bacteria. The lowest MIC values for bacteria and fungi were obtained for ‘Grosso’. Furthermore, the ‘Grosso’ oil showed the highest fungicidal activity, while the highest bactericidal activity was found in ‘Hidcote Blue’ and ‘Grosso’. Using Staphylococcus aureus as an example, it was shown that different bacterial strains of the same species show varying sensitivity to the essential oil. A higher oil content was noted for the cultivars L. × intermedia, especially for the ‘Phenomenal’. Linalyl acetate and linalool were the main components of the essential oil in all cultivars. However, in the ‘Grosso’ oil, a high content of terpinen-4-ol (18.08%) was also recorded. An analysis of the relationships between the content of the main components in the analysed essential oils and the antimicrobial activity of essential oils suggested that linalool and terpinen-4-ol were compounds potentially responsible for antimicrobial activity. The obtained results allow us to conclude that essential oil with significant antimicrobial activity can be obtained from Lavandula sp. plants harvested in the northern part of the cultivation range.