Entrepreneurial ecosystems have become a hotspot of research because they offer a comprehensive understanding of the entrepreneurial context. In this context, incubators, as intermediary players, are critical to promoting new business creation and enabling ecosystem activity and economic growth. To reach this goal, ecosystem members—particularly business incubator managers—seek ecosystem strategies, including cooperation and competition. While previous literature indicates that successful ecosystems require a balance between cooperation and competition to maintain the improvement of value creation, little evidence exists of the effects on performance. Therefore, this article empirically tests the effects of the entrepreneurial ecosystem and co-opetition strategy on incubator performance. The literature on ecosystem strategy and strategic fit is followed to conceptualize the links among the entrepreneurial ecosystem, co-opetition strategy, and incubator performance. An online quantitative survey-based method was used to examine the strategic interactions in the entrepreneurial ecosystem. The empirical analysis of data from 156 incubators in France revealed that co-opetition significantly improves incubator performance and that the entrepreneurial ecosystem has a moderating role between incubator strategy and performance. Additionally, this article suggests that policymakers may favor hybrid strategies such as co-opetition over pure strategies such as cooperation or competition under certain conditions. This evidence supports the implementation of holistic strategies in relation to the organization’s external environment.