Lyme disease diagnostic workups conducted on active and retired U.S. service members and their dependents at U.S. Air Force military treatment facilities (MTFs) in Germany between 2013 and 2017 were assessed to determine the appropriateness of laboratory testing and antibiotic prescriptions. Of the 1,176 first-tier immunoassays, 1,114 (94.7%) were negative, and of the 285 immunoglobulin M (IgM) immunoblots, 242 (84.9%) followed a negative first-tier assay or were performed without an antecedent first-tier assay. Eighty-three positive IgM immunoblot tests were adjudicated using modified published criteria, of which 40 (48.2%) were deemed false positives. Thirtytwo patients with false-positive tests were treated with an antibiotic. Additionally, 30 patients with uncomplicated erythema migrans could have been treated without laboratory confirmation. Understanding the use and limitations of 2-tier diagnostic criteria, as well as the common pitfalls in diagnosing Lyme disease, may help prevent overdiagnosis, reduce unnecessary testing, and promote antibiotic stewardship.