In the quest for new antibacterial agents, a series of novel long‐ and medium‐chain mono‐ and disubstituted β‐lactones was developed. Their activity against three pathogenic mycobacteria—M. abscessus, M. marinum, and M. tuberculosis—was assessed by the resazurin microtiter assay (REMA). Among the 16 β‐lactones synthesized, only 3‐hexadecyloxetan‐2‐one (VM005) exhibited promising activity against M. abscessus, whereas most of the β‐lactones showed interesting activities against M. marinum, similar to that of the classical antibiotic, isoniazid. Regarding M. tuberculosis, six compounds were found to be active against this mycobacterium, with β‐lactone VM008 [trans‐(Z)‐3‐(hexadec‐7‐en‐1‐yl)‐4‐propyloxetan‐2‐one] being the best growth inhibitor. The promising antibacterial activities of the best compounds in this series suggest that these molecules may serve as leads for the development of much more efficient antimycobacterial agents.