The present study provides a comprehensive survey of the planktonic hydrozoan fauna from the Paranaguá Estuarine System (PES; southern Brazil, 25°S 48°W), a subtropical estuary considered a Natural World Heritage site by UNESCO. Extensive collections were performed throughout the estuary in five sampling campaigns during the summer and winter periods of 2012 and 2013 and summer of 2014, totalling 185 samples. About 49,000 organisms were analysed which together with the few previous records resulted in a total of 36 hydromedusae and three siphonophore species. We highlight the presence of Cnidostoma fallax in high abundance (>19,000 individuals captured; ~40% of all planktonic hydrozoans). The high abundance of this species throughout three consecutive summers suggests a change in the local assemblage structure, since previous reports in the 1980s, 1990s and 2000s had shown Liriope tetraphylla recurrently as the dominant species which now ranks fourth. It is difficult to ascertain the causes of such changes due to the paucity of previous studies on Brazilian estuaries, but one possibility is that C. fallax has been recently introduced. In any case, the high dominance of this hydromedusa was not expected and this scenario would potentially cause changes in the local food web since C. fallax and L. tetraphylla are quite different morphologically and ecologically and thus probably play different trophic roles. Future studies are necessary to check the long-term permanency of C. fallax and to assess its biology and ecology and the impact of this assemblage change on the ecosystem.