The mass production of antibiotics in the 1940s enabled their travel beyond Europe and America, but to date the significance of the ways in which these medicines co-constituted colonial regimes at the time has not been systematically described. Through a case study of yaws and syphilis, this research article traces arrivals of antibiotics in three countries of Eastern Africa—Malawi, Zimbabwe, and Uganda. We draw attention to the emergent roles of antibiotics at the intersection of colonial governance and humanitarianism in these different settings. Through this analysis of archival and ethnographic materials, we explore how antibiotics became ‘infrastructural’ in material, affective, and political ways. Achieving a better understanding of the entanglement of antibiotics with human systems and lives is crucial to address the pressing issue of antimicrobial resistance (AMR). With this article we join in the global multidisciplinary efforts to tackle AMR, pointing out the often-overlooked role of colonial history in the circulation of antibiotic drugs, and opening a line of research that will provide valuable insights for the development of effective measures to prevent and reduce the spread of antibiotic resistance.