Molecular tools have revealed wide microbial diversity in the human alimentary tract. Most intestinal microorganisms have not been cultured and the in situ functions of distinct groups of the intestinal microbiota are largely unknown but pivotal to understanding the role of these microorganisms in health and disease. Promising strategies to gain more insight into the functionality of the complex microbial communities in the human alimentary tract, including fermentation processes in the colon, are discussed. These research approaches could provide a basis for the definition of a healthy gut based on key properties of microbial functionality. This will also enable the development of direct nutritional strategies for intestinal disease prevention and health promotion.