Background: The microbiological basis of diaper dermatitis is not clearly elucidated, although a better knowledge of microbial colonisation can be of importance with regard to an adequate treatment. Objective: To investigate the relevance of Candida sp. and Staphylococcus aureus colonisation in diaper dermatitis and to determine the correlation between the extent of colonisation and the severity of disease. Methods: Growth of Candida sp. and S. aureus in the perianal, inguinal and oral regions was determined by positive/negative and semi-quantitative analysis in an open, multi-centre (n = 3) study. Forty-eight children with healthy skin and 28 with diaper dermatitis were analysed. The severity of diaper dermatitis was assessed using a total symptoms score. Results: Colonisation by Candida sp. was significantly more frequent in children with diaper dermatitis as compared to those with healthy skin (perianal 75 vs. 19%; inguinal 50 vs. 10%; oral 68 vs. 25%, p < 0.0003), whereas colonisation by S. aureus at the 3 swab locations was not different (p > 0.34). There was a highly significant, positive correlation between severity of disease and extent of Candida sp. colonisation at all swab locations. Conclusions: Limited microbial colonisation in diaper dermatitis is of questionable relevance, but extensive colonisation seems to aggravate the symptoms; therefore, we suggest that semi-quantitative evaluation should be preferred to the positive/negative assessment for a differential diagnosis.