Biosecurity seems to be the most promising tool for Campylobacter control on poultry farms. A longitudinal molecular epidemiological study was performed during two production cycles, in which the broilers, the poultry house, and the environment of 10 (mixed) broiler farms were monitored weekly. Cecal droppings from the second production cycle were also used for 16S metabarcoding to study the differences in the microbiota of colonized and uncolonized flocks. Results showed that 3 out of 10 farms were positive for Campylobacter in the first production cycle, and 4 out of 10 were positive in the second. Broilers became colonized at the earliest when they were four weeks old. The majority of the flocks (57%) became colonized after partial depopulation. Before colonization of the flocks, Campylobacter was rarely detected in the environment, but it was frequently isolated from cattle and swine. Although these animals appeared to be consistent carriers of Campylobacter, molecular typing revealed that they were not the source of flock colonization. In accordance with previous reports, this study suggests that partial depopulation appears to be an important risk factor for Campylobacter introduction into the broiler house. Metabarcoding indicated that two Campylobacter-free flocks carried high relative abundances of Megamonas in their ceca, suggesting potential competition with Campylobacter.