Fascioliasis is a zoonosis actually considered as a foodborne trematode disease priority by the World Health Organization. Our study presents three cases of F. hepatica infection diagnosed by direct, indirect and/or imaging diagnostic techniques, showing the need of the combined use of them. In order to overcome some difficulties of the presently available methods we show for the first time the application of molecular tools to improve human fascioliasis diagnosis by employing a PCR protocol based on a repetitive element as target sequence. In conclusion, diagnosis of human fascioliasis has to be carried out by the combination of diagnostic techniques that allow the detection of infection in different disease phases, different epidemiological situations and known/new transmission patterns in the actual scenario.