Prevalence of tick-borne disease and emergence of novel tick-borne pathogens have been increasing in North America. Numerous abiotic and biotic variables can affect tick populations and the relative influence of these variables on ticks can differ across scales. We determined the seasonal phenology for questing ticks in three regions of the southeastern United States, assessed relative importance of select habitat and microclimate variables on tick abundance and presence, and determined the prevalence of six different pathogens in the ticks collected. We observed spring activity peaks for adults and nymphs of Amblyomma americanum, and winter activity peaks for adult Ixodes scapularis. Ticks were associated with sites with a deeper litter layer and denser canopy closure. Pathogen prevalence in our ticks was consistent with previously reported results in the literature. These data provide additional information on tick phenology, habitat drivers, and pathogen prevalence across a broad geographical range of the southeastern US.