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Background: The AquaCHROMTM ECC method from CHROMagar is intended for the detection and enumeration of Escherichia coli and coliform bacteria in 100 mL water samples after 18–24 h of incubation at 35–37C. Objective: To validate the AquaCHROM ECC method for qualitative and quantitative detection of E. coli and coliforms with different water matrixes. Methods: Inclusivity/exclusivity studies were conducted. AquaCHROM ECC was compared to U.S. cultural reference methods in unpaired matrix studies for detection of E. coli and coliforms in tap water, well water, lake water, and bottled water, and for enumeration in tap water, well water, and lake water. Three production lots of AquaCHROM ECC were tested for product consistency and stability. Variations in incubation time and temperature were evaluated in robustness testing. Results: Inclusivity/exclusivity results demonstrated expected performance with the exception of three strains of Salmonella enterica, two species of Shigella, and one strain of Aeromonas, which turned the media blue or yellow. Results from the matrix studies demonstrated that AquaCHROM ECC and the reference methods are not statistically different for detection of E. coli and coliforms and statistically equivalent for enumeration of E. coli and coliforms. The AquaCHROM ECC powder production was proven to be consistent with a 24-month shelf life. Variation in temperature did not affect the method performance. Shortening the incubation time is not recommended. Conclusion: AquaCHROM ECC is an effective method for the detection and enumeration of E. coli and coliforms for the water matrixes evaluated. Highlights: The AquaCHROM ECC method is a quick, one-step method for the recovery and enumeration of E. coli and coliforms in 100 mL water samples. It is a non-agar-based chromogenic medium which provides a clear result without the use of a UV lamp. [ABSTRACT FROM AUTHOR]