Environmental DNA (eDNA) water samples were collected at 15 tree islands containing wading bird breeding colonies (order Pelecaniformes) and 15 empty control islands in the central Everglades of Florida in spring of 2017 (April through June) and analyzed for the presence of eDNA from invasive Burmese pythons (Python bivittatus). The Burmese python is now established as a breeding population throughout south Florida, USA. Pythons can consume large quantities of prey and may be a particular threat to wading bird breeding colonies in the Everglades. To quantify python occupancy rates at tree islands where wading birds breed, we utilized environmental DNA (eDNA) analysis—a genetic tool which detects shed DNA in water samples and provides high detection probabilities compared to traditional survey methods. We fitted multi-scale Bayesian occupancy models to test the prediction that Burmese pythons occupy islands with wading bird colonies in the central Everglades at higher rates compared to representative control islands in the same region containing no breeding birds.