Objective: To investigate the antimicrobial and antioxidative effect of the essential oil (EO) of immortelle [Helichrysum italicum (Roth) G. Don (H. italicum)] and its ability to inhibit biofilm of nontuberculous mycobacteria, M. avium and M. intracellulare. Materials and methods: a broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the H. italicum EO. Survival of mycobacteria in Middlebrook 7H9 broth under the influence of H. italicum EO was monitored by subcultivation of suspension of 0, 1, 4 and 8 days. For determination of the inhibition of biofilm formation in water, the biofilm coloring method with crystal violet was applied. The antioxidant effect of MIC concentrations of H. italicum EO was determined by DPPH (2,2-diphenyl-1-picrylhydrazil) method. Results: MIC and MBC value of immortelle EO was 3.2 mg/ml. Treatment of mycobacteria with H. italicum EO at one half times the MIC resulted in approximately 2 log10 reduction of M. avium and 2.5 log10 M. intracellulare respectively over 24 h, and complete reduction of both mycobacteria on the fourth day. M. avium showed a greater tendency to form biofilms. The concentration of one-quarter and one-half of the MIC values resulted in statistically significant inhibition of biofilm production of the tested mycobacteria. MIC concentrations of H. italicum EU after 60 minutes inhibited 38.2% of DPPH radicals. Conclusion: The subinhibitory concentrations of H. italicum EO significantly reduce the viability of M. avium and M. intracellulare and result in substantial inhibition of biofilm production of the tested mycobacteria in water, which would allow the application of low effective concentrations of this EO that do not have a cytotoxic effect as a natural disinfectant in water.