Objectives: Non-tuberculous mycobacteria are opportunistic pathogens that cause disease mainly in im-munocompromised hosts. The present study assessed the prevalence of antibiotic resistance among such mycobacteria from domestic and wild animals in Croatia sampled during several years within a national surveillance program.Methods: A total of 44 isolates belonging to nine slow-growing species were genotyped and analyzed for susceptibility to 13 antimicrobials often used to treat non-tuberculous mycobacterial infections in humans.Results: Most prevalent resistance was to moxifloxacin (77.3%), doxycycline (76.9%), and rifampicin (76.9%), followed by ciprofloxacin (65.4%), trimethoprim-sulfamethoxazole (65.4%), and linezolid (61.4%). Few isolates were resistant to rifabutin (7.7%) or amikacin (6.8%). None of the isolates was resistant to clarithromycin. Nearly all isolates (86.4%) were resistant to multiple antibiotics.Conclusion: Our findings suggest substantial risk that human populations may experience zoonotic in-fections with non-tuberculous mycobacteria that will be difficult to treat using the current generation of antibiotics. Future work should clarify how resistance emerges in wild populations of non-tuberculous mycobacteria.& COPY; 2023 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )