Investigation of major viruses responsible for acute viral gastroenteritis, such as norovirus (NoV), rotavirus species A (RVA) and human adenovirus (HAdV), was conducted in the mountainous region of the state of Rio de Janeiro in a lettuce-producing area. Irrigation water and lettuce samples were collected at different production stages. Viruses were concentrated using an adsorption-elution method and detected by quantitative polymerase chain reaction (qPCR). We detected HAdV in all collection points, although no virus infectivity was shown. The RVA was the most prevalent virus from both water (16.7% [10/60]) and lettuce samples (11.1% [4/36]), with loads ranging from 2.97 × 102 to 6.88 × 103 genomic copies per litre (gc L-1) and 6.24 × 102 to 1.30 × 104 gc per 25 g, respectively. NoV was detected in 8.33% [8/96] in water and lettuce samples, with concentrations ranging from 7.29 × 101 to 1.92 × 103 gc L-1 and from 4.29 × 101 to 2.98 × 103 gc 25 g-1, respectively. Escherichia coli values also demonstrated poor quality of the irrigation and washing water. The presence of at least two different virus strains in all sites reveals the need to improve basic sanitation measures in order to increase food safety.