The initial, rapid expansion of the invasive round goby (Neogobius melanostomus) throughout the Great Lakes drainage was largely confined to lentic systems. We recently observed round gobies ascending two tributaries of the St. Lawrence River. The expansion of gobies into small lotic environments may place ecologically similar species at risk. Fantail darter (Etheostoma flabellare) is one of the several benthic species of the New York Great Lakes drainages that are threatened by round goby invasion. We examined the habitat use and diet composition of fantail darters and round gobies in Mullet Creek, a third-order tributary of the St. Lawrence River, NY, USA. The objectives of this study were to determine the degree of habitat and diet overlap between fantail darters and round gobies in a tributary of the St. Lawrence River. Gobies and darters co-occurred at 22% of capture sites. Of the four habitat variables examined (cover, depth, substrate and velocity), only depth use was significantly different with gobies using deeper habitats than darters. Among the two species and size classes sampled (large vs. small), large darters had the most restricted habitat use requirements. There was variation in round goby and darter diet composition, but only moderate diet overlap occurred between fantail darters and round gobies (Cλ = 0.43). Conditions in Mullet Creek were appropriate for the evaluation of possible spatial and dietary competition between round goby and native darters. Early detection and management of round goby invasions is critical to maintaining ecological integrity of lotic ecosystems in the St. Lawrence Valley.