This study investigated the occurrence of Escherichia coli, an indicator of fecal contamination, in saliva samples from 141 children up to 36 months old, 53 not attending and 88 attending a child daycare center after exposure to a public daycare center and home environments. Two samples from each child were collected on the same day at 07:00 and 15:00 h and plated on MacConkey agar for identification. Samples E. coli negative in the morning and positive in the afternoon were statistically associated with the condition of the child attending daycare center (Odds ratio = 2.72; 95% confidence interval = 1.15/6.46). Exposure to the daycare center environment favored the potential risk of transmission of enteropathogens, as demonstrated by the presence of E. coli in saliva. The method proved to be easy to sample, non-invasive and feasible in young children. The findings suggest a novel and potentially valuable exposure measure.