Fracking fluids were released into Acorn Fork, KY, a designated Outstanding State Resource Water, and habitat for the threatened Chrosomus cumberlandensis (Blackside Dace). As a result, stream pH dropped to 5.6 and stream conductivity increased to 35,000 μS/cm, and aquatic invertebrates and fish were killed or distressed. The objective of this study was to describe post-fracking water quality in Acorn Fork and evaluate if the changes in water quality could have extirpated Blackside Dace populations. Semotilus atromaculatus (Creek Chub) and Lepomis cyanellus (Green Sunfish) were collected from Acorn Fork a month after fracking in lieu of unavailable Blackside Dace. Tissues were histologically analyzed for indicators of stress and percent of fish with lesions. Fish exposed to affected Acorn Fork waters showed general signs of stress and had a higher incidence of gill lesions than unexposed reference fish. Gill lesions observed were consistent with exposure to low pH and toxic concentrations of heavy metals. Gill uptake of aluminum and iron was demonstrated at sites with correspondingly high concentrations of these metals. The abrupt and persistent changes in post-fracking water quality resulted in toxic conditions that could have been deleterious to Blackside Dace health and survival.