High host biodiversity is hypothesized to dilute the risk of vector-borne diseases if many host species are ‘dead ends' that cannot effectively transmit the disease and low-diversity areas tend to be dominated by competent host species. However, many studies on biodiversity–disease relationships characterize host biodiversity at single, local spatial scales, which complicates efforts to forecast disease risk if associations between host biodiversity and disease change with spatial scale. Here, our objective is to evaluate the spatial scaling of relationships between host biodiversity and Borrelia (the bacterial taxon which causes Lyme disease) infection prevalence in small mammals. We compared the associations between infection prevalence and small mammal host diversity for local communities (individual plots) and metacommunities (multiple plots aggregated within a landscape) sampled by the National Ecological Observatory Network (NEON), an emerging continental-scale environmental monitoring program with a hierarchical sampling design. We applied a multispecies, spatially-stratified capture–recapture model to a trapping dataset to estimate five small mammal biodiversity metrics, which we used to predict infection status for a subset of trapped individuals. We found that relationships between Borrelia infection prevalence and biodiversity did indeed vary when biodiversity was quantified at different spatial scales but that these scaling behaviors were idiosyncratic among the five biodiversity metrics. For example, species richness of local communities showed a negative (dilution) effect on infection prevalence, while species richness of the small mammal metacommunity showed a positive (amplification) effect on infection prevalence. Our modeling approach can inform future analyses as data from similar monitoring programs accumulate and become increasingly available through time. Our results indicate that a focus on single spatial scales when assessing the influence of biodiversity on disease risk provides an incomplete picture of the complexity of disease dynamics in ecosystems.