Acadia National Park (ANP) is a non-continuous conglomerate of park lands interspersed with private lands, which is located mainly on Mount Desert Island, ME. Surveys of fishes in ANP have largely ignored or under-sampled most of the streams on the island. Reported here are the results of a 2009 survey that sampled freshwater fishes in 43 streams flowing through Park boundaries. Overall, 19 species were collected, and many were recorded from specific streams for the first time. Notemigonus crysoleucas (Golden Shiner) was the most widely distributed species (47% of surveyed streams), while Pimephales promelas (Fathead Minnow) was recorded from ANP for the first time as a recent bait-bucket introduction. This survey provides an updated baseline for ANP stream fishes and will be used to identify streams for restoration and measure the impacts of future environmental perturbations.