The results of integrated human and veterinary surveillance for West Nile virus (WNV) infections in Austria during the transmission seasons 2015 and 2016 are shown. Altogether WNV nucleic acid was detected in 21 humans, horses, wild birds and mosquito pools. In detail: in four human clinical cases [two cases of West Nile fever (WNF) and two cases of West Nile neuroinvasive disease (WNND)]; eight blood donors [among 145,541 tested donations], of which three remained asymptomatic and five subsequently developed mild WNF; two horses with WNND, of which one recovered and one had to be euthanized; two wild birds [one goshawk and one falcon, both succumbed to WNND]; and five Culex pipiens mosquito pools. Compared to previous years the number of infections increased remarkably. All infections were recorded in the city of Vienna and neighboring regions of Lower Austria. Sixteen coding-complete WNV sequences were established which were closely related to each other and to other Austrian, Czech and Italian viruses, all belonging to the Central/Southern European cluster of WNV sublineage 2d. However, several genetically slightly different WNV strains seem to co-circulate in the same area, as demonstrated by phylogenetic analysis. Based on detailed sequence analysis, all newly discovered Austrian WNV strains had the potential to cause neurological disease, but no correlation was found between severity of disease and the analyzed genetic virulence/neuroinvasiveness markers. Results of integrated human-animal-vector surveillance presented in this paper provide a comprehensive description of WNV activity in the region and will facilitate proactive public health measures to prevent or mitigate potential outbreaks.