Human adenoviruses (hAdVs) of subgroup F (enteric serotypes 40 and 41) display characteristic gut tropism, in vivo, fastidious growth characteristics in cell culture, and are estimated to be associated with 5-20% worldwide of acute gastroenteritis cases among infants and young children. Adequate hAdV gastroenteritis case management requires laboratory-based diagnosis. The present study aimed to the development and evaluation of 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 environmental and/or clinical samples, since no LAMP assay has previously been reported for the detection of these virus types. The assay targeted the hexon gene and had the advantages of being rapid, simple, specific, and sensitive. Results could be obtained within 60 min, under isothermal conditions at 69 °C. The detection limits for hAdV genomes were between 50 and 100 copies/reaction for hAdV40 and hAdV41, and no cross-reactions with other selected viruses, were found. The assay was evaluated with clinical as well as environmental samples. The developed assay is expected to provide a potential molecular tool in obtaining greater knowledge of the hAdV40/41 importance in the epidemiology and clinical manifestations of gastroenteritis.