This study assesses the risks in ecological restoration arising from transplanting into soil containing glyphosate residues. Four Australian restoration species were grown for 60 days in nonadsorbing media treated continuously with glyphosate to establish threshold concentrations for damage. Visual signs of injury were observed in three species, and severe effects on root growth in all species, at solution concentrations as low as 18 mg/L. Only the perennial grass Themeda sp. died at this concentration, with other species surviving at concentrations in the range 36–360 mg/L, beyond which all plants died. Fourteen days exposure followed by removal of glyphosate from root media produced similar effects. Field and glasshouse experiments with the relatively tolerant tree species Angophora costata showed that application rates in the range 10–50 L/ha of herbicide product (360 g/L) would be needed to sustain damage to young plants transplanted into soil typical of local restoration sites. The volume of spray delivered using a hand-operated sprayer varied between operators by 5- and 10-fold to complete the same tasks, at the high end presenting a potential risk to the most tolerant species under field conditions, even when spray concentrations follow label instructions. For all but the most sensitive species, the risk of glyphosate residues in ecological restoration should be minimized by training operators of unregulated applicators to deliver controlled volumes of herbicide when spot spraying prior to transplanting.