While European innovation ecosystems are facing increasing global competition, there are big differences between European regions’ innovative capacity. Funding obstacles, lack of investors, and scarcity of acceleration services are especially prevalent in less connected innovation ecosystems in Eastern and Southern Europe. This represents a significant hurdle to unleashing the potential of European entrepreneurship. As an answer to these challenges the SPREAD2INNO project aimed to understand the needs of startups and SMEs in various innovation ecosystems, focusing on financial and business services. Through the implementation of a three-phase startup acceleration programme, the project contributed to enhancing business activities in less developed innovation regions in Bulgaria, Greece and Italy. Connecting regional startups with local stakeholders helped to increase innovation, promote inclusion and diversity, as well as evaluate the sustainability of the methodology for long-term impact. Additionally, an online platform was created to provide a single point of reference for information exchange between different innovation ecosystems about opportunities, events and funding accessible in these regions. As a result, the SPREAD2INNO project contributed to boosting innovation ecosystems by enhancing their openness, competitiveness, and global reach while creating high-value local jobs, attracting investments, and enabling startups and SMEs to access services and networks across Europe for greater internationalization.