The role of bacteria in the causation of sudden infant death syndrome (SIDS) is gaining acceptance. Mainstream research favouring respiratory compromise has failed to provide a plausible pathogenetic mechanism despite many years of investigation and thousands of research papers. Bacterial colonisation of the colon of the human infant is influenced by many factors including age, mode of delivery, diet, environment, and antibiotic exposure. The gut microbiome influences development of the immune system. The gut microflora could be important in protection against the bacteria and/or their toxins purportedly involved in SIDS pathogenesis. The aim was to perform a preliminary investigation of the gut microflora in sudden infant death syndrome (SIDS) compared with live comparison babies. The intestinal contents from 52 SIDS, and 102 faecal samples from age-matched live comparison infants were screened by PCR to target 16s RNA genes of Clostridium innocuum, Cl. Perfringens, Cl. difficile, Bacteroides thetaiotaomicron and Staphylococcus aureus. Gut colonisation of the babies with these bacteria was analysed in relation to age, gender and type of feeding; and for SIDS babies sleeping position. Cl. difficile, Cl. innocuum and B. thetaiotaomicron were significantly associated with SIDS with 25%, 46% and 30% of cases PCR positive for these respective bacteria compared with only 6%, 23% and 8.8% respectively in the comparison group. SIDS babies had dual colonisation by both Cl. perfringens and Cl. difficile significantly more often than comparison babies and also with triple colonisation by Cl. perfringens, Cl. difficile and Cl. innocuum. SIDS babies were more often colonised by S. aureus than comparison babies. In addition, SIDS babies found prone were significantly more likely to be colonised by S. aureus than for other positions recorded (OR = ∞; CI = 2·04 - ∞). No significant differences between breast and bottle-fed SIDS babies was observed in regard to each clostridial bacterium, or S. aureus, however Cl. innocuum was found to be significantly associated with formula feeding in the comparison cohort. Comparison of breast and formula feeding of SIDS babies with live comparison babies revealed significant differences with regards to some of the clostridial bacteria. Age-specific differences in gut bacterial microbiome were observed in both SIDS and comparison healthy babies. This study gives an insight into differences in the gut bacterial microbiome of SIDS babies compared with healthy babies. These differences could be important in contributing to a baby's susceptibility to infection and therefore to SIDS. The association of S. aureus colonisation with prone sleep position supports the hypothesis that prone sleep position could increase the risk of ingestion/inhalation of bacteria contaminating the sleeping surface and could account for the increased risk of SIDS in babies who are put to sleep prone. The study provides impetus for broader studies into the gut microbiome of babies and could lead to effective approaches to SIDS prevention.