The acquisition of reduced nitrogen (N) is essential for plant life, and plants have developed numerous strategies and symbioses with soil microorganisms to acquire this form of N. The evolutionary history of specific symbiotic relationships of plants with soil bacteria, however, lacks evidence from the fossil record confirming these mutualistic relationships. Here we use modern plants in the N-fixing clade of rosids to develop a geochemical method to assess the presence of symbiotic relationships with N-fixing soil bacteria via δ15N values of tree rings. Application of this method to Oligocene tree rings confirms the symbiosis of certain arborescent legumes with N-fixing soil bacteria. The results suggest actinorhizal symbiosis for some Oligocene non-leguminous trees. The specific age, genera, and presence or absence of bacterial symbiosis of these fossil trees provide new information on genera that have maintained or lost the ability to form symbioses in the N-fixing clade. We envision that this approach, as applied to paleoecology, can lead to greater understanding of the response of plant symbioses under variations in atmospheric chemistry for N-limited ecosystems.