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Escherichia coli is a member of the normal microbiota of the gut of mammals and due to its metabolic versatility it can survive in natural ecosystems. The survival of E. coli in aquatic ecosystems requires this microorganism to overcome environmental stress. Although multiple investigations are carried out to assess the prevalence of E. coli in the environment as an indicator of fecal contamination, few focus on the study of the phenotypic characteristics of the environmental strains of this important microorganism. The objective of this research was to determine the biochemical profiles of E. coli strains isolated from aquatic environments with conventional tests and the Entero Well D–One system. In total, 61 isolates of E. coli were collected from September 2017 to January 2018 from water samples of the Almendares, Quibú and Luyanó rivers of Havana. A total of 90.1% of isolates identified had agreement between Entero Well D-One and conventional techniques for identification at genus and species level. Twenty seven different biochemical profiles were obtained between the E. coli strains. The 63.6% presented atypical profiles due to the negative reaction of the biochemical tests like production of the β-D-galactosidase enzyme, decarboxylation of the aminoacids lysine and ornithine and use of sorbitol. The presence of E. coli strains with atypical biochemical profiles shows that E. coli pathotypes could be found in the waters of the rivers of Havana and therefore the use of these waters implies a hygienic-sanitary risk. [ABSTRACT FROM AUTHOR]