The main stem of Darby Creek is designed as a Coldwater Fishery and a Migratory Fishery from its source to the Pennsylvania Route 3 bridge. From that point downstream to its mouth, the non-tidal portions of the creek's main stem are a Trout Stocked Fishery and a Migratory Fishery. Numerous fish species were observed in the creek during a 2009 electrofishing survey by the Pennsylvania Fish and Boat Commission. The catch per unit effort per hour rate for smallmouth bass was slightly over 10 and the rate for rock bass was under 10. The catch per unit effort per hour rate for redbreast sunfish was 123, the highest of several warmwater streams tested during the survey. Some stocked rainbow trout and brown trout were also captured in the creek. High numbers of American eels were observed in the creek was well. Fish species such as mummichogs, silvery minnows, striped bass, and white perch were found at the head-of-tide of the creek. Brown trout naturally reproduce in the creek's headwaters.