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The tick microbiota may influence the colonization of Ixodes scapularis by Borrelia burgdorferi , the Lyme disease bacterium. Using conserved and pathogen-specific primers we performed a cross-kingdom analysis of bacterial, fungal, protistan and archaeal communities of I. scapularis nymphs (N = 105) collected from southern Vermont, USA. The bacterial community was dominated by a Rickettsia and several environmental taxa commonly reported in I. scapularis , as well as the human pathogens B. burgdorferi and Anaplasma phagocytophilum , agent of human granulocytic anaplasmosis. With the fungal primer set we detected primarily plant- and litter-associated taxa and >18% of sequences were Malassezia , a fungal genus associated with mammalian skin. Two 18S rRNA gene primer sets, intended to target protistan communities, returned mostly Ixodes DNA as well as the wildlife pathogen Babesia odocoilei (7% of samples), a Gregarines species (14%) and a Spirurida nematode (18%). Data from pathogen-specific and conserved primers were consistent in terms of prevalence and identification. We measured B. burgdorferi presence/absence and load and found that bacterial beta diversity varied based on B. burgdorferi presence/absence. Load was weakly associated with bacterial community composition. We identified taxa associated with B. burgdorferi infection that should be evaluated for their role in vector colonization by pathogens. [ABSTRACT FROM AUTHOR]