This paper presents the finding of the possible cause of the high false-positive rate in acid-fast staining in histological examinations. Using acid-fast staining, culture, and PCR, acid-fast bacilli were detected in 83.7% of 49 hospital tap water samples and nontuberculous mycobacteria (NTM) were detected in 20.4% of the same 49 samples. The 10 NTM isolates were also identified to the species level using PCR-restriction fragment length polymorphism. Our findings indicate that NTM in hospital tap water are the possible cause of false positives in acid-fast staining and of nosocomial infection in immunocompromised patients.