The presence of IgG and IgA antibodies to Simkania negevensis in adult Italian patients with respiratory or gastrointestinal symptoms was investigated by the microimmunofluorescence test. In patients with respiratory infections, IgG (50%) and IgA (13%) seropositivity was consistent with previous data. In patients with gastrointestinal disorders, IgG (68%) and IgA (18%) seroprevalence was significantly higher than in healthy controls. These results, in association with the previously described detection of S. negevensis in water sources, could suggest an oral route of infection other than droplets or close contact, and a possible association of S. negevensis with gastrointestinal infections.