The emergence of non-tuberculous mycobacteria (NTM) infections, in Europe and North America, is higher than that of M. tuberculosis (M. tb). NTM are ubiquitous and opportunistic in persons with immunodeficiency or chronic lung disease. Among six NTM with proven pulmonary pathogenicity, M. avium complex (MAC) and M. abscessus are the most common. Current NTM treatments are moderately efficient, as they were initially designed to cure M. tb infections. First-line treatments require the combination of at least three antibiotics with different mechanisms of action to limit cross-resistance over a long period (12 to 24 months). Consequently, it is urgent to develop new anti-NTM molecules more specific and more efficient to reduce treatment duration and overcome resistant strains. Two quinoline-based compounds, bedaquiline (BQ) and mefloquine (MQ) target the ATP synthase, a vital enzyme for mycobacteria. However, BQ is only used as a treatment of last resort due to many drug interactions and significant hepatic and cardiac side effects. MQ is safer than BQ but is moderately active against NTM (e.g., MIC = 4 µg/mL on MAC). Recently, we developed a first series of MQ analogs active against MAC and M. abscessus with a better selectivity index (SI) than MQ (SI = 2.86 vs 0.38). In continuation of this work, new MQ analogs with piperazine core were designed, prepared by short asymmetric synthesis and characterized. The first in vitro antimycobacterial evaluations on several strains and cytotoxicity study were carried out.