Early childhood caries (ECC) is closely related to high numbers of mutans streptococci, lactobacilli and Candida albicans. Oral colonization of these microorganisms was monitored in a prospective clinical study in order to investigate the effect of comprehensive treatment under general anesthesia and the sustainability of microbial changes. Saliva samples were collected from 50 healthy infants with ECC before and in regular intervals up to 12 months after treatment. Microorganisms were detected by cultivation on selective agars (CRT® bacteria and Sabouraud/CandiSelect™) and scored. Additionally, plaque on upper front teeth and the dmft were recorded. Parents were repeatedly interviewed regarding the children's diet and oral hygiene, accompanied by corresponding advice. Plaque frequency and the numbers of mutans streptococci, lactobacilli and yeasts were significantly reduced as a result of treatment (p < 0.0001, Wilcoxon test). Nevertheless, this effect was not permanent. An ordinal regression model on the follow-up period revealed that the odds for bacteria and yeasts to reach a higher score increased linearly over time (p < 0.01) with an odds ratio of 2.244 per year. One third (34%) of the children developed new dentinal lesions within 1 year postoperatively. High scores of lactobacilli before treatment predicted caries relapse (p < 0.05). Nutritional and oral hygiene habits changed only slightly despite advising. Elimination and restoration of ECC lesions under general anesthesia proved to be an effective procedure in reducing cariogenic bacteria and yeasts. A satisfactory and sustainable success, however, could be achieved neither regarding microbiologic parameters nor with respect to the relapse rate. More suitable strategies are needed.