Groundwater hydrophobic acids isolated from the Tomago Sand Beds, Australia have been structurally characterised using multiple analytical techniques. Temporal variability was observed during the study period (1996–2003) and data for two representative samples isolated in 1996 and 2002 are presented. The sample isolated in 2002 was a highly aromatic fulvic acid with branched aliphatic functional structures and high oxygen content. It differed from structural concepts which suggest that groundwater hydrophobic acids isolated from organically lean aquifers are predominantly aliphatic in nature, with a low oxygen content resulting from extended degradation processes. Data are consistent with the hydrophobic acids having inputs from both surface recharge and mineralised sedimentary organic carbon. The sample isolated in 1996 was significantly more aliphatic, indicating that a change in microbial activity/community has occurred at the site.