Clostridium botulinum causes infant botulism by colonising the intestines and producing botulinum neurotoxin in situ. Previous reports have linked infant botulism cases to C. botulinum spores in household dust, yet the baseline incidence of C. botulinum spores in residential households is currently unknown. Vacuum cleaner dust from 963 households in 13 major Canadian cities was tested for C. botulinum using a novel real-time PCR assay directed against all known subtypes of the botulinum neurotoxin gene. None of the samples tested positive for C. botulinum. Analysis of a random subset of samples by MALDI Biotyper revealed that the most common anaerobic bacterial isolates were of the genus Clostridium and the most common species recovered overall was Clostridium perfringens. Dust that was spiked with C. botulinum spores of each toxin type successfully produced positive real-time PCR reactions. These control experiments indicate that this is a viable method for the detection of C. botulinum spores in household dust. We make several recommendations for future work that may help discover a common environmental source of C. botulinum spores that could lead to effective preventative measures for this rare but deadly childhood disease.