The detection of pathogenic microorganisms in aquatic environments is extremely relevant in terms of public health. As these laboratorial methodologies are usually difficult, expensive and time-consuming, they are frequently replaced by the assessment of fecal indicator bacteria, such as Escherichia coli. This study aimed to assess the presence of E. coli in fecal samples from Neotropical otters, to evaluate its potential as fecal indicator to be applied to the determination of water microbiological quality in areas where otters' populations are high. Twenty-six otter fecal samples, collected in Alto Paranapanema river basin, São Paulo State, Brazil, were analyzed for the presence of E. coli, using conventional bacteriological methods. Only 8 scat samples (30%) were E. coli positive, indicating that this microorganism is not a suitable fecal indicator to assess water fecal contamination by Neotropical otters, and should not be used to infer the presence of otter related pathogens in waters.