Summary Despite a strong uptake of evidence‐based approaches, conservation often proceeds from a grossly incomplete understanding of species priorities. To optimize conservation impact within a biogeographical region, quantitative knowledge is needed of the species present, which should be prioritized, and the management interventions these require. The next challenge is to avoid a proliferation of competing species plans, or conversely, a lack of detail within generic habitat‐based approaches. We present a methodology for biodiversity auditing. We quantified regional biodiversity by systematically collating available species records, allowing objective prioritization. We collated autecological information to integrate multiple species into management guilds with shared requirements, providing evidence‐based guidance for regional conservation. For two regions of Eastern England, Breckland (2300 km2) and The Broads (2000 km2), we collated 0·83 and 1·5‐million records, respectively. Numbers of species (12 845 and 11 067) and priority species (rare, threatened, designated or regionally restricted: 2097 and 1519, respectively) were orders of magnitude greater than previously recognized. Regional specialists, with a UK range largely or entirely restricted to the region, were poorly recognized posing a risk of regional homogenization. A large body of autecological information existed for priority species and collating this allowed us to define cross‐taxa management guilds. Numbers of priority species requiring different combinations of ecological processes and conditions were not matched by current conservation practice in Breckland. For example, the current agri‐environment agreements for designated grass heaths potentially catered for only 15% of the 542 priority species and 21% of 47 regional specialists that could potentially benefit from evidence‐based management. A focus on vegetation composition rather than the ecological requirements of priority species underpinned this failure. Synthesis and applications. The biodiversity audit approach provides an objective model for prioritization and cost‐effective conservation, applicable to regions of Europe where biodiversity has been well characterized. By using this approach to collate available information, management guilds with similar requirements can be defined across taxa, providing evidence‐based guidance for regional conservation.