Invasive species are a major driver in the global decline of biodiversity. Invasive herpetofauna cause ecological harm through different mechanisms that vary in scope and severity, and Florida boasts more established nonnative herpetofauna species than any other region in the world. There, black spiny-tailed iguanas (Ctenosaura similis) are one of several large invasive reptiles known to occupy the burrows of the imperiled, native gopher tortoise (Gopherus polyphemus) and may even exclude tortoises from their burrows. To test the hypothesis that iguanas exclude tortoises from their burrows, we conducted a field study on Gasparilla Island, Florida, USA. We used a burrow scope to estimate occupancy of each species within tortoise burrows at sites with and without sustained iguana removal efforts and modelled co-occurrence patterns between the two species. We used two-species occupancy analyses to test three predictions relating to gopher tortoise burrow use. Our results support the hypothesis that gopher tortoises are excluded from their burrows by black spiny-tailed iguanas. The energetic cost to a tortoise of excavating a new burrow is unknown but may be substantial. In addition, tortoises are more vulnerable to extreme temperatures and predation while searching for a new burrow location and digging a burrow. Our results also show that sustained iguana removal is likely effective at reducing iguana occupancy of tortoise burrows. Other large invasive reptiles and perhaps even some native species typically thought of as “burrow commensals” may have similar deleterious effects on tortoise behavior.