Invasive Burmese pythons have been shown to have population-level effects on native mammals in southern Florida. Tens of thousands of long-legged wading birds (of multiple species in Ciconiiformes, Pelecaniformes) breed in aggregations, known as colonies, on tree islands in the Everglades. Burmese pythons may pose a threat to these colonies because pythons are semi-aquatic and commonly use tree islands and arboreal habitat. However, python predation on nests of wading birds has not previously been documented or quantified. We used trail cameras to monitor nests at colonies in Everglades National Park and Water Conservation Area 3 in 2014, and 2016–2017. We did not detect Burmese python predation at monitored nests in 2014 (23 nests in 2 colonies) or 2016 (59 nests in 4 colonies). In 2017 (125 nests in 7 colonies), we detected three individual pythons consuming nestlings, fledglings, and eggs in a minimum of 7.9% (5 nests, n = 63) of monitored nests at a colony in Everglades National Park. In 2017, the overall predation rate of Burmese pythons at all monitored nests (5 of 125 nests, or 4%), was five times the native predator rate (1 of 125 nests, or 0.8%). Our study confirms that Burmese pythons are acting as predators in wading bird colonies at nontrivial rates and provides a baseline to which future studies can refer.