Mycobacterium avium complex-related diseases are often associated with poorly maintained hot water systems. This calls for the development of new control strategies. The aim of this study was to investigate the activity of essential oils (EOs) from the Mediterranean plants, common juniper, immortelle, sage, lavandin, laurel, and white cedar against Mycobacterium avium ssp. avium, Mycobacterium intracellulare, and Mycobacterium gordonae in culturing broth and freshwater as their most common habitat. To do that, we developed a new method of water microdilution to determine their minimal effective concentrations (MEC). The most active EO was the one from the common juniper with the MEC of 1.6 mg mL-1. Gas chromatography / mass spectrometry the juniper EO identified monoterpenes (70.54 %) and sesquiterpenes (25.9 %) as dominant component groups. The main monoterpene hydrocarbons were α-pinene, sabinene, and β-pinene. The juniper EO significantly reduced the cell viability of M. intracellulare and M. gordonae at MEC, and of M. avium at 2xMEC. Microscopic analysis confirmed its inhibitory effect by revealing significant morphological changes in the cell membrane and cytoplasm of all three bacteria. The mode of action of the juniper EO on the cell membrane was confirmed by a marked leakage of intracellular material. Juniper EO has a great practical potential as a complementary or alternative water disinfectant in hot water systems such as baths, swimming pools, spa pools, hot tubs, or even foot baths/whirlpools.