Microbiological control of hospital waters as one of the main sources of nontuberculous mycobacteria (NTM) is important for the prevention of NTM-associated illness. This study aimed to investigate the prevalence of NTM in the hospital water systems of Tehran, Iran. A total of 218 samples from different hospital waters (i.e., tap water and medical devices such as humidifying cup of oxygen manometer, dialysis devices, nebulizers, and dental units) were included in this study. Phenotypic and molecular tests were used to identify the isolated organisms to species level. Of 218, 85 (39.0%) samples at 37 °C and 87 (40.0%) samples at 25 °C were identified as NTM. Using hsp65-sequencing method, Mycobacterium lentiflavum was the most frequently encountered, followed by M. gordonae and M. paragordonae. No significant difference was seen in frequency and species in mycobacteria isolated at 37 °C and 25 °C temperatures. Humidifying cup of oxygen manometer had the most contaminated water among the investigated water distribution systems in hospitals. Isolation of NTM from hospital water sources is a serious public health problem in Iran and merits further attention by health authorities. Establishment of microbiological monitoring systems for hospital waters and expanding the number of facilitated laboratories are strongly recommended.