Invasive alien species (IAS) constitute a trans-and intracontinental challenge. With this article, we exemplify obstacles met during the implementation of the Regulation (EU) No. 1143/2014 against IAS and comparatively assess them through two case studies from two Member States, Austria and Romania, via qualitative content analysis. Challenges are identified, analyzed, and discussed based on interviews with stakeholders from both countries in the view of the three main intervention types within this Regulation: prevention, early detection, and rapid eradication and management. The obstacles, which the two Member States met, are related to creating, updating, and maintaining the Union List and to the distribution of competences, which influence the national and subnational cooperation and coordination. Financial resources, custom controls, national legislation, and awareness influence the implementation of the IAS Regulation too. This research contributes to improving knowledge about numerous details of the implementation and enforcement of this IAS-Regulation to make them more feasible, not just for scientists, but also for decisionand policymakers, business owners, and other stakeholders.