National Public Health Institutes (NPHIs) around the world vary in composition. Consolidated organizational models can bring together critical functions such as disease surveillance, emergency preparedness and response, public health research, workforce development and laboratory diagnosis within a single focal point. This can lead to enhanced coordination and management of resources and enable more efficient and effective public health operations. We explored stakeholders’ perceptions about the benefits and challenges of consolidating public health functions in an NPHI in seven countries where the US Centers for Disease Control and Prevention has supported NPHI establishment and strengthening. From August 2019 through January 2020, we interviewed a total of 96 stakeholders, including NPHI staff (N = 43), non-NPHI government staff (N = 29) and non-governmental and international organization staff (N = 24) in Cambodia, Colombia, Liberia, Mozambique, Nigeria, Rwanda and Zambia. We conducted a policy analysis using Tea Collins’s health policy analysis framework to assess various possible options for coordinating public health functions and their likely effectiveness. The findings can be used by policymakers as they consider public health infrastructure. We found that consolidating functions in an NPHI, to the extent politically and organizationally feasible, promotes efficiency, flexibility and coordination, as well as supports data-driven health recommendations to government decision makers. Countries pursuing NPHI establishment can weigh the potential challenges and benefits of consolidating functions when determining which public health functions will comprise the NPHI, including clarity of role, access to resources, influence over decisions and political viability.