Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that can result in clinical and subclinical infection in many species. It is also considered the cause of a potential zoonotic disease. In the present study, a serological survey was conducted using samples from pet dogs in the state of Florida. Twenty-seven of 125 serum samples, or 21.6%, were found to demonstrate reactivity by ELISA, with titers ranging from 1:32 to 1:512. There were no significant differences by age or sex. This is the first report on the detection of E. cuniculi antibodies in dogs in the United States.