Ixodes scapularis, the blacklegged tick, is capable of transmitting the pathogens that cause Lyme disease (Borrelia burgdorferi), babesiosis (Babesia microti), anaplasmosis (Anaplasma phagocytophilum), and to a lesser extent Powassan encephalitis (deer tick virus [DTV]). These pathogens represent significant public health problems, but little is known about the occurrence and co-infection prevalence of these pathogens in I. scapularis. Here, we used standard PCR and pathogen-specific primers to estimate the prevalence of infection of A. phagocytophilium, B. burgdorferi, B. microti, and Ehrlichia chaffeensis in questing nymph and adult I. scapularis collected from sites in Putnam and Dutchess counties in southern New York in 2011. To detect DTV infection, cell cultures were observed for the presence of cytopathic effects and positive results were confirmed via real time RT-PCR. In 466 individually sampled adult ticks, B. burgdorferi had the highest prevalence of infection (55%) followed by A. phagocytophilum (18.2%), DTV (3.4%), B. microti (3.2%), and E. chaffeensis (1.5%). Infection with two pathogens occurred in 13.3% of ticks, and 10 ticks were infected with three combinations of three pathogens. These results provide an estimate of the rate of co-infection, which then can help inform the epidemiological risk of contracting multiple zoonotic tick-borne pathogens within the Hudson Valley region of New York State.