Woody biomass production can increase through establishment of non-native tree species exhibiting greater growth potential than traditional native species. Interest in growing Eucalyptus in the southeastern US has raised concern over its potential spread and invasion, which could impact ecosystem properties and functions. Within the matrix of land use where Eucalyptus establishment is being considered in the southeastern US, surrounding pine forests managed with fire represent a likely pathway for invasion. We used greenhouse and field experiments to evaluate the potential invasion risk of Eucalyptus benthamii. We were specifically interested in determining if seeds could successfully germinate in fire-maintained pine forests and if fire-return intervals influenced germination through impacts on litter accumulation and light availability. The greenhouse experiment investigated the influence of light availability on germination success, whereas the field study investigated the influence of time since fire, and thus litter accumulation and light availability, on germination success. Percent germination was similar under non-shaded controls and moderate shade, but complete shade resulted in low germination rates. Germination was lower in the field compared to the greenhouse and was influenced by litter and light availability, which varied according to fire-return intervals. Litter increased, and light availability decreased, with time since burn. Germination was negatively related to litter depth and positively related to light availability, thereby decreasing with time since fire. Germination increased with litter removal but remained positively related to light availability after litter removal. Higher germination with litter removal suggests germination is influenced by litter, but higher germination with increased light availability, regardless of raking, suggests germination is also influenced by light availability. Despite these relationships, no seedlings persisted through the growing season. The low germination rates under a variety of field conditions coupled with the lack of persistence suggests establishment may be unlikely, regardless of the surrounding land matrix.