Antimicrobial resistance (AMR) is a rising global concern, exacerbated by inappropriate antimicrobial use (AMU) in agriculture, which accelerates resistance in pathogens affecting both humans and animals. Addressing AMR requires coordinated strategies, with the One Health Approach (OHA) emphasizing integrated actions across human, animal, and environmental sectors. This study assessed institutional capacity for AMU and AMR control through the OHA in the Kilimanjaro and Mwanza regions of Tanzania. Six districts were purposively selected to capture diverse economic activities, population densities, and AMU practices, while ensuring contextual variation. A qualitative design was employed, using key informant interviews and focus group discussions with stakeholders from health, veterinary, and environmental sectors. Data were coded using both deductive and inductive approaches, with validation through double-coding, peer debriefing, and triangulation of sources. Findings revealed institutional gaps, including weak enforcement of AMU regulations, limited veterinary support, inadequate laboratory capacity, and insufficient intersectoral collaboration. Farmers and officers emphasized the risks of counterfeit drugs, poor-quality products, and misuse of human antimicrobials in livestock production. While limited to two regions, the study’s robustness was strengthened by methodological rigor and alignment with regional evidence. Beyond confirming existing challenges, the findings advance global AMR discourse by showing how institutional weaknesses manifest differently across socio-economic and ecological contexts, highlighting the need for context-sensitive operationalization of the OHA. Strengthening institutional capacity, improving veterinary and human health services, and fostering meaningful community engagement are critical to achieving Tanzania’s National Action Plan on AMR (2023–2028) and contributing to global AMR control efforts