Background Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult Nontuberculous Mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6 %. Thus, novel treatment drugs are needed, of which we identified the drug Clomiphene Citrate (CC), which treats infertility in women, to exhibit inhibitory activity against M. abscessus. Methods To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. Results CC significantly inhibited the growth of not only wild-type M. abscessus strains, but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC’s drug-efficacy did not have a significant cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. Conclusions The Results of this in vitro study on M. abscessus activity suggest that CC is a potential new drug for the treatment of M. abscessus infections.