Babesia microti is a protozoan that infects red blood cells, causing hemolytic anemia and flu-like symptoms in humans. Understanding co-infections is crucial for the better diagnosis, treatment, and management of tick-borne diseases. This study examined the prevalence of Babesia microti co-infection with other prevalent tick-borne pathogens in Pennsylvania. The dataset acquired from the Dr. Jane Huffman Wildlife Genetics Institute included passive surveillance data from Ixodes spp. from 2021 to 2023. Submitted ticks were screened for tick-borne pathogens using species-specific TaqMan qPCR. Of the 793 B. microti-positive ticks pulled for analysis, 65.0% were co-infected with other pathogens (n = 516). Notably, 60.9% of the B. microti-positive ticks were co-infected with Borrelia burgdorferi, 10.2% with Anaplasma phagocytophilum Ap-ha, and 7.5% carried a triple co-infection with B. burgdorferi and A. phagocytophilum Ap-ha. The rates of B. microti infection and its co-infections are on the rise, with patterns observed in Pennsylvania and other regions of the USA. While other studies have collected both nymphal and adult ticks to screen for co-infections in Pennsylvania, our study stood out as a unique contribution to the field by focusing exclusively on B. microti-positive ticks. The continued monitoring of tick-borne co-infections is vital to prevent misdiagnosis and ensure effective treatment regimens.