A High-throughput Method (HTM) for processing Sponge-Stick Samples to detect Bacillus anthracis - spore-forming biothreat agent for anthrax - was recently developed. The current dataset is for a manuscript which describes how the High-throughput Method (HTM) developed for the spore-forming biothreat agent was adapted and evaluated for the non-spore forming biothreat agents, Yersinia pestis (causes Plague) and Francisella tularensis (causes Tularemia). This dataset is associated with the following publication: Brisson, V., S. Kane, M. Calfee, S. Cendrowski, and S. Shah. Evaluation of a High-Throughput Processing Method for Sponge-Stick Samples to Detect Viable, Non-Spore-Forming Biothreat Agents. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 236: 107194, (2025).