Knowledge and understanding about how the Earth functions and supports life create the foundation for ecological literacy. Industrialisation, urbanisation and population growth have resulted in changed relationships between many human communities and the natural world. A potential consequence is a compromised capability to make well-informed decisions about how to live sustainably. To gain a measure of ecological literacy within the South Australian community, we collaborated with senior scientists and educators to develop and apply an instrument with the capacity to determine indicative levels of ecological knowledge and understanding. A formal, variable credit, multiple-choice assessment instrument was distributed online to groups and individuals within diverse community sectors and industries. Quantitative analyses of scores indicated that levels of ecological knowledge and understanding within a self-selected sample of over one thousand individuals ranged from very low to extremely high, with the majority of respondents achieving moderate to high scores. This instrument has a demonstrated capacity to determine indicative levels of ecological literacy within and between individuals and groups. It is able to capture mastery of ecological knowledge and understanding achieved through both formal and informal pathways. Using the results, we have been able to establish a range of standards and an aspirational target score for the South Australian community. The value of this work is in its potential to deliver insights into relationships between humans and the rest of the natural world, and into characteristics of eco-literate individuals and communities, that might not otherwise emerge.