It is well known that watershed-based source water protection programs are integral to the provision of clean drinking water. However, the involvement of Indigenous communities in these programs is very limited in Canada, which has contributed to the vulnerability of Indigenous source waters to contamination. Through a partnership with an Anishinaabe community, this research aimed to identify challenges and opportunities for communities and practitioners to improve the protection of Indigenous source waters in the province of Ontario. The methodology followed the Indigenous research principles of relationship, respect, relevance, reciprocity, and responsibility. Interviews and a youth focus group were conducted with Indigenous community members and practitioners from industry, academia, non-governmental organizations (NGOs), and government. Analysis was conducted using an iterative process to develop codes and themes in the qualitative data analysis software NVivo. Results indicated that issues with scale, jurisdiction, the concept of source water protection, representation, funding, and capacity impact efforts to protect Indigenous source waters. Hopeful recent developments and upcoming opportunities were identified, and a water protection framework for First Nation communities in Ontario was developed in partnership with an Anishinaabe water protection committee. Recommendations are provided to multiple sectors for moving forward respectfully, and effectively, towards the protection of Indigenous waters.