The Mary River turtle (Elusor macrurus) is an endemic, monotypic species with multiple impacts across its life-history, including overharvesting of eggs, nest predation and habitat degradation. Long-term recruitment failure has led to protection measures established under state, federal and international authority. Previous research has demonstrated that E. macrurus lives instream but nests on river banks, requiring specific habitat for breeding, nesting and recruitment. Ecohydrological rules represent the critical water requirements contributing to a species’ life history and can be used to develop and assess environmental flow strategies for species affected by water resource development. This study investigated the nesting behaviour of E. macrurus, including the environmental drivers that affect nest inundation. Monitoring showed that nesting by E. macrurus peaked in October and November, driven by rainfall events (>10 mm), with potential impacts from flow events (20% of nests established <2.5 m above water level at time of nesting). These ecohydrological rules were modelled against 109 years of simulated natural flow and rainfall data. The ‘potential nesting and nest inundation’ (PNNI) indicator revealed that nesting for E. macrurus was assured in a majority of years under the natural flow scenario. The results of this study will inform the development and assessment of e-flow strategies for nesting by E. macrurus in terms of current, and future water resource development, along with climate change impacts.