Between December 2012 and May 2017, we conducted a fungal inventory at the Boston Harbor Islands National Recreation Area (BHI) in Massachusetts. We extensively sampled 4 sites (Grape Island, Peddocks Island, Thompson Island, and World's End peninsula) and occasionally visited 4 others for sampling (Calf Island, Great Brewster Island, Slate Island, and Webb Memorial State Park). We made over 900 collections, of which 313 have been identified. The survey yielded 172 species in 123 genera, 62 families, 24 orders, 11 classes, and 2 phyla. We report 4 species as new, but not formally described, in the genera Orbilia, Resupinatus, and Xylaria. Another collection in the genus Lactarius may be new to science, but further morphological and molecular work is needed to confirm this conclusion. Additionally, Orbilia aprilis is a new report for North America, Proliferodiscus earoleucus represents only the second report for the US, and Chrysosporium sulfureum, a common fungus of some cheeses, was discovered on woodlice (Crustacea: Malacostraca: Isopoda: Oniscidea). We discuss our findings in the light of DNA-based identifications using the ITS ribosomal DNA region, including the advantages and disadvantages of this approach, and stress the need for biodiversity studies in urbanized areas during all seasons.