Between September 6 and 21, 2012, seven human cases of West Nile virus (WNV) neuroinvasive infection were laboratory confirmed in Croatia. The median patient age was 62 years (range 48-77). Five patients presented with meningoencephalitis and two patients with meningoencephalitis followed by acute flaccid paralysis. Four of them had an underlying disease (hypertension). Using enzyme-linked immunosorbent assay (ELISA), WNV-specific immunoglobulin M (IgM) and IgG antibodies of low avidity were detected in six patients, whereas one showed only IgM antibodies. All samples were confirmed using plaque-reduction neutralization and microneutralization tests. Five patients recovered fully. Before human cases were reported, acute asymptomatic WNV infection was demonstrated by detection of IgM antibodies in sentinel horses. Moreover, an increased WNV IgG seropositivity in horses was detected in counties where human cases occurred. Adulticidal and larvicidal treatments were administered immediately in the respective places of residence. The end of the warm season contributed to the fact that there were no new cases of WNV disease recorded.