This study aimed to survey the environmental dissemination of enterovirus (EV) in a site of organic lettuce situated in the mountainous region of the state of Rio de Janeiro, Brazil. For this purpose, a total of 96 environmental samples, including water and lettuce samples obtained in different stages of the production chain (e.g., irrigation water, seedlings, lettuces grown, and washed lettuces ready-to-eat), were analyzed. EV genomes were detected in 12.5% (12/96) of the tested samples (eight from irrigation water and 4 from lettuce samples). Levels of viral concentration ranged from 3.37 × 103 to 4.72 × 106 genomic copies per liter (gc L-1 ) and from 2.14 × 104 to 5.56 × 104 genome copies per 25 grams (gc 25 g-1 ) for the water and lettuce samples, respectively. Such findings suggest that the use of viruses as human fecal contamination markers must be considered in order to improve food safety in organic supply chains.