Strongyloides stercoralis is a parasite that can cause death in immunocompromised people. A proper diagnosis is hence essential. The real-time polymerase-chain reaction (RT–PCR) is a novel, promising diagnostic method, that detects the DNA of the parasite in stool samples. In this retrospective study, we compared the sensitivity of agar plate coproculture (APC), an in-house immunofluorescence test (IFAT) and an in-house RT–PCR for the diagnosis of S. stercoralis infection. The study sample was composed by 223 samples. Samples resulting positive to APC, IFAT and RT–PCR were 20, 140 and 25, respectively. When sensitivity was calculated against a composite reference standard, serology confirmed the best performance (sensitivity 95%), followed by RT–PCR (57%) and APC (45%). In conclusion, in a non-endemic setting, serology is the best screening method, while the combination of APC and RT–PCR does not seem a reasonable approach to increase sensitivity. Both methods can have a role as confirmatory tests for selected cases.