Purpose We report on first evidence of simultaneous occurrence of West Nile virus (WNV) and Usutu virus (USUV) neuroinvasive infection in humans in Croatia during the transmission season 2013. Methods From June to December 2013, a total of 95 patients with clinically suspected WNV infection (WNV fever and neuroinvasive disease) were tested for WNV IgM/IgG antibodies using enzyme-linked immunosorbent assay. Twenty-six reactive samples were further tested by virus neutralization test for confirmation. Results WNV neuroinvasive infection was confirmed in 20 patients, while in three patients USUV neutralizing antibodies were detected. Cases occurred during the 11-week interval (from 24 July to 07 October 2013). Both WNV and USUV cases were distributed in three north-western Croatian counties. In addition to human cases, recent asymptomatic WNV infection (detection of IgM antibodies) was recorded in 9/3,460 (0.3 %) tested sentinel horses. Infected animals were recorded in two eastern and one north-western county. Conclusions Our results indicate co-circulation of WNV and USUV in Croatia. WNV infection could be misdiagnosed with other emerging infectious diseases presenting with neurological symptoms such as USUV infection.