Plasmodium falciparum is a significant human pathogen, particularly in the historical context of the ancient Mediterranean region. The causative species of malaria are “invisible” in the historical record, while malaria as a disease entity is indirectly supported by evidence from literary works (e.g., the Hippocratic Corpus, Celsus’ De Medicina) and non-specific skeletal pathological responses. Although ancient DNA may demonstrate the presence of a pathogen, there remain theoretical and methodological challenges in contextualizing such molecular evidence. Here we present a framework to explore the biosocial context of malaria in 1st–4th c. CE central-southern Italy using genomic, literary, epidemiological, and archaeological evidence to highlight relationships between the Plasmodium parasite, human hosts, Anopheles vector, and environment. By systematically integrating these evidentiary sources, our approach highlights the importance of disease ecology (e.g., climate and landscape) and human-environment interactions (e.g., land use patterns, such as agriculture or infrastructure activities) that differentially impact the potential scope of malaria in the past.