Introduction: The prevalence of tick-borne diseases is poorly understood in Franche-Comté. The primary objective of this first regional study was to estimate the incidence of Lyme disease and to identify the presence of six other tick-borne diseases: tick-borne encephalitis, human granulocytic anaplasmosis, TIBOLA (tick-borne lymphadenopathy), LAR (lymphangitis-associated rickettsiosis), tularemia, and Q fever. Materials and Methods: From April 1, 2010, to March 31, 2012, a network of physicians (general practitioners and specialists) reported all newly diagnosed cases of these diseases to the Regional Intervention Unit (CIRE) of Santé publique France. Case validation and classification were performed by a committee of experts for Lyme borreliosis using a case definition based on a European consensus (European Concerted Action on Lyme Borreliosis—EUCALB) and, for the other diseases, according to criteria defined by the National Reference Centers. Results: The participation rate among physicians was 12%, consistent with other studies of this type. Of the 604 reports, 432 (71%) cases of Lyme borreliosis were included. Nearly 91% of the included patients presented with erythema migrans. No reports of the six other tick-borne diseases were validated. The number of new cases of Lyme borreliosis was estimated at 2,000 cases over 24 months, bringing the average annual incidence rate to 85 cases per 100,000 inhabitants (95% CI: [77–92]). Incidence rates varied by department and canton. Discussion-Conclusion: Franche-Comté has an incidence rate of the same order of magnitude as that estimated by the Sentinelles network in 2009–2012. This rate, higher than that estimated for mainland France (42 per 100,000 inhabitants, 95% CI: [37–48]), ranks the region third after Limousin and Alsace for 2009–2012.