Offshore wind is an emerging sector in Australia, yet the risks and potential impacts that offshore wind farm (OWF) development may pose for Little penguins (Eudyptula minor) remain poorly understood. As part of the Australian Government Department of Climate Change, Energy, the Environment and Water’s Renewables Environmental Research Initiative, this project synthesized current knowledge of Little penguin biology through a targeted literature and spatial data review, with a focus on describing regional patterns and potential impact pathways associated with OWF developments. Our synthesis highlights substantial regional variation in colony size, population trends, habitat availability, movement ecology, and the timing of key life cycle stages across regions, which may influence sensitivity to impact pathways. Using this ecological evidence, we identified key knowledge gaps and developed regulatory guidance and survey guidelines for addressing interactions between Little penguins and OWF development under the EPBC Act. By converting evidence into applied guidance, this project illustrates how marine science can support informed policy, planning, and long-term conservation outcomes in Australia’s changing marine environment.