The ability of viruses to persist in the environment and on fresh produce, as well as their low infectious doses, allows even a small amount of contamination to cause foodborne viral outbreaks. Human Adenoviruses (hAdVs) of subgroup F (enteric serotypes 40 and 41) are known to be associated with 5–20% worldwide of acute gastroenteritis cases among infants and young children. The present study aimed to evaluate a simple and cost-effective, one-step, single-tube adenovirus type 40/41 specific loop-mediated isothermal amplification (LAMP) assay for the detection of hAdV40/41 DNA in a series of ready-to-eat food samples (strawberries, sour cherries, lettuce, cherry tomatoes and green onions). No LAMP assay has previously been reported for the detection of these virus types in food samples. Results were obtained within 60 min, under isothermal conditions at 69°C. After amplification, the products were detected either by observing a ladder pattern following gel electrophoresis, or a color change with the addition of SYBR Green I to the reaction tube. The limits of detection of hAdV40/41 in food samples were found to be >30 GCs per reaction, when a nucleic acids extraction step was included in the analysis. A 1 log higher (>100 GCs/reaction) detection limit was found for lettuce, cherry tomatoes and green onions, without a nucleic acids extraction step included before the isothermal amplification. The LAMP assay for the virological analysis of food samples is expected to provide a robust, innovative, powerful, cheap and fast monitoring tool, without the need of sophisticated equipment, which will be available for food safety testing by the food industry and the public food health authorities.