This study was carried out to evaluate the antibacterial activity of aqueous and organic extracts of Thymus vulgaris L. (Lamiaceae). The hexan (HEX), ethanol (ETH) and water (WAT) extracts from aerial parts of T. vulgaris exhibited antimicrobial activity against slow growth mycobacteria – SGM (n = 49) and rapid growth mycobacteria – RGM (n = 50) strains isolated from diseased ornamental fish. The MICAV values of HEX, ETH, and WAT extracts against RGM strains were found to be at 146.50 µg/mL, 728.00 µg/mL, and > 800 µg/mL, respectively. The MICAV values of HEX, ETH, and WAT extracts against SGM strains were found to be at 50.77 µg/mL, 253.06 µg/mL, and > 800 µg/mL, respectively. The most active extract against RGM and SGM strains was the hexane extract. Its chemical composition was determined using GC-MS, which allowed the identification of 53 compounds. The major chemical compounds in HEX extracts were found to be thymol (27.35%), tetratetracontane (19.68%), dotriacontane (10.34%), p-cymene (5.92%), stigmast-5-en-3-ol (4.89%), carvacrol (2.64%), thymoquinone (2.00%), (1r)-1,6,6-trimethyl-cis-bicyclo[3.3.0]octan-3-one (2.52%), 5,10-dihexyl-5,10-dihydro indole[3,2b]indole-2,7-dicarbaldehyde (2.15%), and linalyl anthranilate (1.06%), the proportion of other components being below 1%. In conclusion, among the tested extracts, the HEX extract exhibits the strongest properties against nontuberculosis mycobacteria isolated from diseased ornamental fish.