Recreational activities can be detrimental to biodiversity; for example, off-road vehicle traffic (e.g., ATV riding), which has become increasingly popular in recent decades, can threaten wildlife. Although ATV riding around wetlands may threaten the shallow nests of turtles, there are no data on the effect of ATVs on turtle nests. We studied nest site choice and nest survival in two species of softshell turtles (Apalone mutica and A. spinifera) along a river in Louisiana before (1993–1994) and after (2015–2016) ATV riding became popular at the site to determine whether ATVs were an important source of nest mortality, and whether there was an effect of nest site choice on nest survival. ATVs were the most common source of nest mortality (one-third of nests destroyed); nest mortality was significantly positively related to increased ATV traffic but was not influenced by species or nest site choice. Experiments with surrogate eggs and an ATV revealed that the most vulnerable nests to ATV mortality were those that were shallower, were driven over more slowly, and were turned upon. We recommend restricting the access of riding clubs to the river; enforcement of regulations on isolated riders from adjacent residential areas will be logistically and financially challenging.