Conservation fences have been used as a tool to stop threatening processes from acting against endangered wildlife, yet little is known of the impacts of fences on non-target native species. In this study, we intensively monitored a pest-exclusion fence for 16 months to assess impacts on a reptile community in south-eastern Australia. We registered 1052 reptile records of six species along the fence. Encounters and mortality were greatest for eastern long-necked turtles (Chelodina longicollis), whereas impacts on lizards (Tiliqua rugosa, Tiliqua scincoides, Pogona barbata, Egernia cunninghami) and snakes (Pseudonajatextilis) were more moderate. We recorded several Chelodina longicollis recaptures at the fence and many of these were later found dead at the fence, indicating persistent attempts to navigate past the fence. We conservatively estimate that the fence resulted in the death of 3.3% and disrupted movements of 20.9% of the turtle population within the enclosure. Movement disruption and high mortality were also observed for turtles attempting to enter the nature reserve, effectively isolating the reserve population from others in the wider landscape. Of 98 turtle mortalities, the most common cause of death was overheating, followed by predation, vehicular collision, and entanglement. Turtle interactions were clustered in areas with more wetlands and less urban development, and temporally correlated with high rainfall and solar radiation, and low temperature. Thus, managers could focus at times and locations to mitigate impacts on turtles. We believe the impact of fences on non-target species is a widespread and unrecognized threat, and suggest that future and on-going conservation fencing projects consider risks to non-target native species, and where possible, apply mitigation strategies that maintain natural movement corridors and minimize mortality risk.