Purpose Inclusion of biodiversity as an indicator in the land use impact pathway of Life Cycle Assessment (LCA) is essential to assess the effects of human activities on the environment. Numerous models have been applied, but validations that use actual data, collected in the field, are scarce. Methods The expert system SALCA-BD (Swiss Agricultural LCA-Biodiversity), assigns coefficients for land use class suitability and impact of agricultural practices on species diversity at field and farm scale. We used data on land use classes and agricultural practices from 132 farms located in eight European regions to complete the life cycle inventory. SALCA-BD species diversity scores were calculated for individual fields, aggregated to the farm scale, and compared to field records of arable crop flora, grassland flora, spiders, and wild bees. Results and discussion Overall, species diversity scores from SALCA-BD were positively related to the observed species richness from field survey data. The extent of the relationship diminished from arable crop flora and grassland flora to spiders and to wild bees, and from field to farm scale. Conclusions Validation of a LCA biodiversity assessment tool with data from field surveys revealed the benefit of considering multiple aspects of biodiversity. The appropriate scale for species diversity assessment (as a proxy for biodiversity) is the respective species habitat. Extension of scale increases uncertainty, which should be addressed by developing characterization factors for as detailed a land use classification as possible.