Human eastern equine encephalitis (EEE) is a life-threatening mosquito-borne disease. To determine whether mosquito abundance and EEE virus infection rates are associated with human EEE disease, we evaluated retrospectively a total of 592,637 mosquitoes and onset dates for 20 confirmed human cases over 26 years in Massachusetts. Annual Culiseta melanura populations at 10 defined sites decreased over the study period (P = 0.002). Weekly infection rates and number of infected Culiseta melanura captured per trap night were positively associated EEE cases (P < 0.023 and P < 0.001, respectively), whereas abundance was not (P = 0.077). The infection rate for Culiseta melanura of 0.39 per 1,000 tested mosquitoes identified human cases with a sensitivity of 0.87, a specificity of 0.82, a positive predictive value of 0.14, and a negative predictive value of 0.995. Timely mosquito testing and infection rate calculation are critical for disease risk estimation and outbreak control efforts.