The health burden of waterborne nontuberculous mycobacteria (NTM) is a rising concern. While the water supply systems can serve as a potential reservoir for NTM, their abundance, diversity, and transmission pathways remain unknown. This study aimed to characterize the prevalence and regrowth of NTM in building water supply system in a hospital where many M. abscessus were isolated from patients. The depletion of residual chlorine after stagnation and supply of warm water at the point of use promoted significant microbial regrowth, including NTM, in the hospital. The absolute abundance of Mycobacterium spp. 16S rRNA genes in tap water and shower water samples increased to approximately 10