Since the beginning of this century, the European Union (EU) has been committed to tackling the continuously accelerating biodiversity crisis. Yet, both its 2010 and 2020 Biodiversity Strategy failed to deliver their main targets of halting and reversing biodiversity loss. The design of the new Biodiversity Strategy for 2030 acknowledges lessons learned from the evaluation of these past strategies. However, improving on the ground implementation of biodiversity protection efforts requires a comprehensive understanding of the challenges occurring on the target-level. Horizon Europe project BioAgora aims to set up the scientific pillar of the European Commission to ratchet up the EU BDS implementation. As first task of BioAgora, this research investigates target-specific challenges of implementing past strategies. By applying a combined methods approach, we identified and categorised a variety of target-specific challenges which, if not addressed, are likely to remain barriers to achieving the 2030 objectives. By revealing those challenges, this typology fundamentally supports ratcheting up the Biodiversity Strategy 2030. We conclude by recommending further, ideally systematic, research on identifying challenges and continuously updating this work throughout the strategy’s timeframe to allow for the impact of adapted governance instruments to be reflected.