The analysis of microorganisms is often not included in environmental groundwater chemistry studies, but an interest in the ecological significance of these organisms is starting to emerge. This is particularly relevant where the transport of excess agricultural contaminants poses a significant risk to the sustainability of important bio-regions like the Great Barrier Reef. This study shows how broad scale agriculture can affect groundwater chemistry and the diversity of groundwater microbiota in the wet tropics of north Queensland, Australia. Eight groundwater monitoring bores were regularly sampled in a sugarcane-dominated catchment for analysis of microbiology and major ion chemistry. Soil samples were also collected from a sugarcane plantation paddock specifically for microbiological analysis. Metagenomic analysis of 16S ribosomal RNA sequences was conducted on extracted biological material from the samples. The results revealed a consortium of abundant bacteria that have associations with a variety of soil, water and plant processes, dominated by Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. These were present in both shallow (<10 m depth) and deep (>35 m depth) groundwater aquifers, suggesting a connectivity between soil and groundwater environments. This shows that, in tropical catchments characterised by high rainfall and heterogeneous alluvium, biological indicators of broad-scale agriculture are not restricted to the upper soil environment. The implications are that anthropogenic activities can exert great control on the biological diversity of aquifer ecosystems.