Introductory biology is a gateway course for majors and other science, technology, engineering, and mathematics (STEM) disciplines. Despite the importance of chemistry content knowledge for understanding biology, the relationship between chemistry knowledge and prior coursework and biology course performance is understudied. We used an opportunity gap framework to investigate the extent to which there were opportunity gaps in prior chemistry coursework and knowledge and associated these gaps with subsequent equity gaps in student performance on introductory biology assessments. We also developed, implemented, and assessed an asynchronous content-based intervention to support student learning and reduce equity gaps. We collected data from ∼1800 students enrolled in seven course sections of introductory biology, including two course sections prior to implementation of the intervention and five course sections with the intervention. We identified opportunity gaps in chemistry coursework that were associated with students' performance on their first introductory biology exam. The results from this study highlight the importance of addressing chemistry concepts early in a course with sufficient support for students and an understanding of opportunity gaps.