Research has shown that the biological and morphological effect of the transition to agriculture varied widely by population and geographical region. In Upper Nubia, the shift to full-scale agriculture included transitional phases with varying dependence on pastoralism and farming, alongside continued hunting and gathering. Therefore, Upper Nubia is an ideal region to study the relationship between subsistence strategy, mandibular morphology and oral health. This study analysed the mandibles and dentition from 102 adult individuals from ancient Nubian populations. The sample contained a Late Palaeolithic hunter-gatherer population from Lower Nubia (c. 13,000–9000 BC), as well as samples representing four cultural periods in Upper Nubia (Sudanese Neolithic c. 5000–4000 cal BC, Kerma Ancien c. 2500–2050 BC, Kerma Classique c. 1750–1500 BC and Meroitic c. 350 BC–AD 350). Mandibular osteometrics and cross-sectional geometric properties were calculated from 3D laser scanned models to explore the relationship between diet-induced biomechanical force and variation in mandibular shape and strength between samples. Dental pathology and wear were used to assess diachronic changes in oral health and dietary composition. Dental size was also measured to compare the relationship between mandibular and dental size over time. The intensive agricultural population from the Kerma Classique period had the highest prevalence and severity of dental pathology and wear, which may reflect a highly cariogenic and abrasive diet. Oral health improved in the subsequent Meroitic sample, possibly due to an increase in dietary heterogeneity and/or improved hygienic practices. Overall, mandibles from the Late Palaeolithic hunter-gatherers were longer, wider, and had more upright and larger rami than subsequent agricultural populations. Mandibular size continued to decrease within the subsequent Upper Nubian samples, most notably in the overall length, body height in the molar region and width of the ramus. The gonial angle also became more obtuse over time. Changes in mandibular size were not accompanied by consistent evidence of dental size reduction. In addition, there were significant differences between the Late Palaeolithic and Upper Nubian samples in mandibular biomechanical properties. Most notably, molar Ix and Imax continued to decrease through agricultural intensification in the Upper Nubian samples, suggesting a reduction in the sagittal bending rigidity of the mandible in response to an increasing reliance on softer agricultural food products. The timing of the mandibular morphological changes indicate that the overall size of the mandible began to decrease before strength relative to size. Overall, this study used a novel combination of methodologies to identify major biological changes in the dentition and mandible during the transition to agriculture in Upper Nubia. Importantly, the results demonstrate that changes in masticatory loading magnitude, as a result of dietary shifts, specifically influence mandibular morphology and robusticity in anatomical regions associated with masticatory function, such as the molar region of the mandibular corpus and the ramus. The findings support the masticatory-functional hypothesis and show that dietary changes are an important factor influencing observed mandibular morphological variation between populations. This study contributes to a better understanding of the biological changes that accompanied the agricultural transition in Ancient Nubia.