Non-tuberculous mycobacteria (NTM) are opportunistic pathogens commonly colonizing hospital water systems, and may be responsible for healthcare-associated infections (HAI). Investigation of HAI and outbreaks caused by NTM necessitates water analyses. However, NTM are slow-growing bacteria within the mesophilic community present in water, and are difficult to detect. Prior to culture on specific media, their recovery usually requires decontamination and concentration steps. We assessed the effectiveness of filtration as regards the recovery of 7 NTM species in hospital water samples. We also compared the use of cetylpyridinium chloride (CPC) at different concentrations and Sodium Hydroxide (NaOH) 4% in decontamination of water samples with mesophilic bacteria. Our laboratory protocol showed that membrane filtration was suitable for concentration and recovery of NTM from water. Sample decontamination with CPC was more NTM-preservative than NaOH. A combination of CPC at 0.005% and filtration allowed detection of NTM at low concentrations, ranging from 3 to 98 CFU/100mL according to the NTM species.