Background: The Bacillus Calmette–Guérin (BCG) vaccine against tuberculosis is the most widespread vaccine in the world. Discovered by French investigators Albert Calmette and Camille Guérin at the Pasteur Institute, it remains the only effective vaccine against tuberculosis infection. This report describes the recognition and identification of a previously unknown French handwritten laboratory notebook prepared by Drs. Calmette and Camille Guérin recording their experiments performed during the development of the BCG vaccine. Methods: The notebook was examined, translated into English, photographed and the experiments analyzed. Results: The laboratory notebook consists of 69 leaves written in 2 hands, one of which corresponds to that of Albert Calmette. This handwritten notebook contains details of experiments that were performed during the development of the BCG vaccine at the Pasteur Institute by Drs. Calmette and Guérin. These include experimental inoculations of rabbits and guinea pigs describing the pathology including skin lesions, inflammatory reactions, organ pathology and survival. The experiments describe varying inoculative dosages of the bacteria, and different routes of administration including intraperitoneal and subcutaneous injections and administration of bacilli in the ear. In those cases where the animal had died following inoculation of tubercle bacilli, necropsy was performed and the organs examined and the pathology findings described. It describes experimental animal deaths and results of necropsies. Conclusions: this previously unknown notebook is a highly organized and detailed record of investigations using tuberculosis in animal experiments and microbiological culture to produce a safe and effective vaccine, first used in humans in 1921.