In this first application to Croatia and Slovenia, the Fish Invasiveness Screening Kit (FISK) version 2 was used to assess the invasiveness potential of 40 introduced and translocated freshwater fish species. Based on a priori classification of invasiveness, ROC analysis of FISK scores from two independent assessors resulted in a statistically significant calibration threshold of 11.75. This indicated that FISK was able to discriminate reliably between non-native species likely to pose a high risk of being invasive and those likely to pose a medium or low risk of invasiveness. Seven species were categorised as ‘medium risk’ and the other 33 as ‘high risk’, whereas no species were categorised as ‘low risk’. The two highest scoring species were European catfish and North African catfish. Mean scores for all species classified a priori as invasive were ranked as ‘high risk’ sensu lato and fell into the ‘moderately high risk’ sub-category. FISK proved to be a useful and viable tool for assessing the risks posed by non-native fishes in Croatia and Slovenia. For this reason, it can be adopted as a reliable tool for the prevention of new translocations or introductions of potentially invasive species in the risk assessment area, as well as to assist in decisions regarding future management (i.e. monitoring, control and eradication) and conservation strategies.