Oxalate nephrosis is a leading disease of the Mount Lofty Ranges koala population in South Australia, but the cause is unclear. In other herbivorous species, a common cause is high dietary oxalate; therefore this study aimed to determine the oxalate content of eucalypt leaves. Juvenile, semimature and mature leaves were collected during spring from eucalypt species eaten by koalas in the Mount Lofty Ranges and compared with those from Moggill, Queensland, where oxalate nephrosis has lower prevalence. Total oxalate was measured as oxalic acid by high-performance liquid chromatography. The oxalate content of eucalypts was low (<1% dry weight), but occasional Mount Lofty leaf samples had oxalate levels of 4.68–7.51% dry weight. Mount Lofty eucalypts were found to be higher in oxalate than those from Queensland (P < 0.001). In conclusion, dietary oxalate in eucalypt leaves is unlikely to be the primary cause of oxalate nephrosis in the Mount Lofty koala population. However, occasional higher oxalate levels could cause oxalate nephrosis in individual koalas or worsen disease in those already affected. Further studies on the seasonal variation of eucalypt leaf oxalate are needed to determine its role in the pathogenesis of oxalate nephrosis in koalas.