Toscana virus (TOSV) is a sandfly-borne phlebovirus causing meningitis and encephalitis during the summer period. A significant proportion of infection results in asymptomatic or pauci-symptomatic forms. Although seroprevalence studies had been conducted in Italy, Spain, Greece, and Cyprus, no data were available from France at the outset of this study. We present here results of seroprevalence studies conducted in volunteer blood donors and in patients presenting with central nervous system (CNS) infections. Twelve percent of sera from blood donors and 18.9% of sera from patients hospitalized for CNS infection contained immunoglobulin G (IgG) reacting against TOSV or TOSV-related phleboviruses. This study confirms that TOSV and possibly TOSV-related phleboviruses actively circulates in southeastern France and demonstrates that a significant proportion of healthy blood donors and patients with CNS infections have a history of TOSV or TOSV-related phlebovirus infection.