Accidental spills of chemicals and other pollutants can decimate populations of stream-dwelling species. Recovery from such accidents can be relatively fast and complete when the affected stream reaches can be recolonized from upstream and downstream sources. However, faunal recoveries from accidental spills that extirpate populations from entire headwater streams have not been extensively documented, and understanding resilience of headwater-stream biota is relevant for assessing threats to at-risk species. We assessed recovery of fish populations in a 5.7-km long headwater stream in the southeastern United States following a complete, or nearly complete, fish-kill caused by a chemical spill near the source of the stream. We sampled for fishes at five stream locations, two downstream and three upstream from a perched, culverted road-crossing located 2.4 km upstream from the stream mouth, over a period of 18.5 months following the poisoning event. We observed 11 fish species, representing <65% of the fish species expected based on occurrences in nearby tributary streams. In post-poisoning sampling, only three of these taxa were observed upstream of the culvert; all 11 species, including the federally threatened Cherokee Darter Etheostoma scotti, were found downstream of the culvert but were mostly represented by a few, large individuals. In contrast, dead individuals of at least eight taxa including the Cherokee Darter were observed upstream of the culvert at the time of the fish-kill. These observations provide evidence of slow recovery of a headwater fish fauna, and especially upstream of a barrier to fish movement, where the recolonization sources are primarily downstream. Additional case studies may reveal whether this result applies generally to headwater streams. Slow recovery could make species that primarily inhabit or maintain greatest abundances in headwaters, including multiple at-risk fishes, particularly vulnerable to the threat of accidental spills that result in local population extirpation.