Research objectives: To investigate the antimicrobial effect of essential oils (EO) of selected Mediterranean plants (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis and Thuja occidentalis) and α-pinene on non-tuberculous mycobacteria (NTM), M. avium, M. intracelullare and M. gordonae and investigate the role of these substances on NTM biofilm. We wanted to determine the synergistic effects of the tested EO and combinations of EO and selected antimicrobials. The aim was also to determine the mechanism of action of common juniper EO on NTM. Methods: The establishment of a new method enabled the in vitro testing of the EO's antimicrobial effect on NTM by mimicking the conditions of water supply systems. The Minimum Effective Concentration (MEC) was determined as the lowest effective EO concentration in water. The sensitivity of NTM to selected EO and antimicrobials was examined by microdilution in broth and sterilized tap water. The effect of inhibitory and synergistic subinhibitory concentrations of common juniper EO and immortelle EO on biofilm of NTM on abiotic and biotic surfaces was investigated. The mechanism of action of common juniper EO has been studied by determining the degree of cell wall damage and morphological changes using a transmission electron microscope (TEM). Results: Common juniper and immortelle EO's proved to be most effective against NTM, while α-pinene tested alone showed poorer effect than overall EO. Common juniper EO have shown a number of synergistic effects, according to NTM, in combination with immortelle and white cedar EO and antimicrobials. The adhesion of NTM to polystyrene as well as biofilm formation were significantly inhibited (p < 0.05) with increasing temperature and increasing concentration of common juniper EO. Synergistic combinations of common juniper EO and antimicrobials have caused increased leakage of NTM cell macromolecules. TEM showed changes in the shape and structure of NTM after the activity of common juniper EO and synergistic effects in combination with immortelle EO. Conclusion: We have found a number of synergistic combinations of EO and antimicrobials that may have potential applications in new ways of preventing the adherence and formation of NTM biofilms not only in the water supply system, but also in artificial materials used in medicine and in biological systems. Synergies with the combined use of EO and antimicrobials allow the use of low effective concentrations with the sustained antimicrobial effect of the test substances with reduced toxicity. Common juniper EO cause changes in the cytoplasm and damage to the NTM cell wall, as evidenced by the observed increased leakage of cellular macromolecules and micromorphological changes observed by TEM.