Aims: A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila, was developed and validated for the detection of H. pylori in environmental samples. Methods and Results: The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori. The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive. Conclusions: This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples. Significance and Impact of the Study: The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.