Eastern oyster (Crassostrea virginica) aquaculture is increasingly playing a significant role in the state of Maine’s (USA) coastal economy. Here, we conducted a qPCR-based survey for Haplosporidium nelsoni, Perkinsus marinus, and Perkinsus chesapeaki in C. virginica (n = 1440) from six Maine sites during the summer–fall of 2016 and 2017. In the absence of reported die-offs, our results indicated the continued presence of the three protozoan parasites in the six sites. The highest H. nelsoni qPCR-prevalence corresponded to Jack’s Point and Prentiss Island ( x ¯ = 40 and 48% respectively), both located in the Damariscotta River Estuary. Jack’s Point, Prentiss Island, New Meadows River, and Weskeag River recorded the highest qPCR-prevalence for P. marinus (32–39%). While the P. marinus qPCR-prevalence differed slightly for the years 2016 and 2017, P. chesapeaki qPCR-prevalence in 2016 was markedly lower than 2017 (<20% at all sites versus >60% at all sites for each of the years, respectively). Mean qPCR-prevalence values for P. chesapeaki over the two-year study were ≥40% for samples from Jack’s Point (49%), Prentiss Island (44%), and New Meadows River (40%). This study highlights that large and sustained surveys for parasitic diseases are fundamental for decision making toward the management of the shellfish aquaculture industry, especially for having a baseline in the case that die-offs occur.