Odocoileus virginianus (white-tailed deer) is the primary host and vector for Ixodes scapularis (deer tick). Most of the research into Ixodes scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the Ixodes populations spreads due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in Ixodes scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer were screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and Powassan virus. Ticks (n = 394) were pooled by sex and life-stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for Powassan virus. This represents a prevalence rate of 0.8% and 1.2%, respectively. Anaplasma phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and Powassan virus detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.