Spores of the bacteria Paenibacillus larvae play a central role in the transmission of American Foulbrood (AFB), a major disease of honey bee (Apis mellifera) colonies. This study investigated whether trained detection dogs could recognise an odour pattern from P. larvae spore samples. Although dogs have previously been used to detect diseased larvae in colonies with AFB, this is the first time they have been investigated for detecting P. larvae spore samples. Given that spores are metabolically inactive, it was unknown whether the spore samples would produce enough volatile organic compounds to form an odour pattern that could be detected by dogs. Three dogs were trained to identify laboratory-produced P. larvae spore samples and were systematically desensitized to non-target odours with a series of control samples. Two of the dogs successfully completed training and were then tested by having each dog perform six searches in an odour-detection carousel with the trainer blinded to the location of the spore samples. In this high-stakes forced-choice test, each dog was asked to identify one new spore sample, containing approximately 93–265 million P. larvae spores, from seven control samples. Both dogs correctly identified the spore sample every time (100% success rate); the probability of this result occurring by chance was p = 0.0000038. Therefore, this study demonstrates that dogs can recognise an odour pattern from bacterial spore samples, in this case, P. larvae, and provides proof of concept for further investigation into the use of detection dogs to reduce the spread of AFB in beekeeping businesses.