This study uses strontium (87Sr/86Sr), oxygen (?18O) and carbon (?13C) isotope analysis of archaeological tooth enamel samples to investigate the origins of human remains from two sites in Arnhem Land, Northern Territory: a coastal Macassan site and an Indigenous rockshelter complex. The study aims to resolve whether two individuals from the Macassan site originate from outside Arnhem Land and, if so, whether their place of origin can be determined. Strontium results confirm the Macassan and Indigenous samples represent two distinct populations. The Indigenous values match the local Arnhem Land geologic strontium signatures, while the Macassan values are outside the local range and more likely to match Indonesian geological signatures. Carbon isotope results are more equivocal, but tend to support the presence of two populations by revealing slightly different dietary backgrounds for each group. Oxygen isotope data introduce more complexity; their geographic signal may be confounded by cultural behaviour. Radiocarbon dating suggests the Macassan Anuru Bay A site is a relatively early contact site. This study shows that even with a small sample set there is potential to discern past human mobility and origin using stable isotope analysis.