Herpes simplex virus (HSV) infections are widespread among humans. Their diagnosis is predominantly clinical, but its laboratory confirmation is increasingly encouraged. We conducted a 6-year retrospective, observational study of polymerase chain reaction (PCR) confirmed mucocutaneous HSV infections in the largest Portuguese tertiary teaching hospital. Throughout this period, a total of 947 PCR herpesvirus tests in mucocutaneous swabs were performed, 448 of which tested positive (266 for HSV). Regarding the PCR-confirmed mucocutaneous HSV infections, HSV-1 was the main cause of orolabial herpes and HSV-2 of genital herpes. A significant proportion of patients were immunocompromised, demonstrating the risk for herpesvirus infection and reactivation in this setting. HSV-1 genital herpes was more common in women, younger individuals and had a lower rate of recurrences. This study demonstrates the variable clinical spectrum of HSV infections, and the importance of their laboratory confirmation, as the recognition of the specific pathogen may determine optimal management and prognosis.