Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to CDI. The main objective of this study was to evaluate and follow the prevalence of C. difficile in a Belgian nursing home. During a 4-month period, stool samples from a group of 23 elderly care home residents were collected weekly. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the faeces content by Targeted Metagenomic analysis. Surfaces and diary meals were also sampled in order to determinate the possible role of environmental and food contamination in the acquisition of CDI. Culture of samples was performed in a selective medium cycloserine cefoxitin fructose cholate. An identification of the isolated colonies was done by PCR detection of tpi, tcdA, tcdB and cdtA genes. Toxic activity was confirmed by a cytotoxic immunoassay. Further characterization was performed by PCR ribotyping. The Metagenomic analysis was targeted on the v1-v3 hyper-variable region of 16S rDNA. The taxonomical assignment of the populations was performed with MOTHUR and Blast algorithms. C. difficile was not detected in any of the tested meals or surfaces samples. For the stools samples, 6 of the 23 controlled residents were identified as C. difficile carriers. The isolates belonged to 4 different PCR ribotypes, including types 020 and 027. This unique association of classical microbiology protocol with pyrosequencing allowed to follow C. difficile in patients and to identify several other bacterial populations whose abundance is correlated with C. difficile.