The introduction and survival of zoonotic bacterial pathogens in poultry farming have been linked to bacterial association with free-living protozoa. To date, however, no information is available on the persistence of protozoan communities in these environments across consecutive rearing cycles and how it is affected by farm- and habitat-specific characteristics and management strategies. We therefore investigated the spatial and temporal dynamics of free-living protozoa in three habitats (pipeline, water, and miscellaneous samples) in three commercial poultry houses across three rearing cycles by using the molecular fingerprinting technique denaturing gradient gel electrophoresis (DGGE). Our study provides strong evidence for the long-term (ca. 6-month) persistence of protozoa in broiler houses across consecutive rearing cycles. Various free-living protozoa (flagellates, ciliates, and amoebae), including known vectors of bacterial pathogens, were observed during the down periods in between rearing cycles. In addition, multivariate analysis and variation partitioning showed that the protozoan community structure in the broiler houses showed almost no change across rearing cycles and remained highly habitat and farm specific. Unlike in natural environments, protozoan communities inside broiler houses are therefore not seasonal. Our results imply that currently used biosecurity measures (cleaning and disinfection) applied during the down periods are not effective against many protozoans and therefore cannot prevent potential cross-contamination of bacterial pathogens via free-living protozoa between rearing cycles.