The presence and colonization of Listeria monocytogenes were investigated in a newly established dairy processing plant during a one-year period. A total of 250 non-food contact surfaces, 163 food contact surfaces, 46 personnel and 77 food samples were analyzed in two different buildings according to the cheese production chain. Initial steps, including salting, are performed in building I (old facility), while the final steps, including ripening, cutting and packaging, are performed in building II (new facility). Overall, 218 samples were collected from building I and 318 from building II. L. monocytogenes isolates were subtyped by PFGE and MLST, and a questionnaire about quality measures was completed. The overall prevalence of L. monocytogenes was 8.40%, and while the presence of the pathogen was observed just during the first sampling in building I, L. monocytogenes was found in building II at the third sampling event. The salting area in building I had the highest proportion of positive samples with the highest diversity of PFGE types. Moreover, L. monocytogenes PFGE type 3 (sequence type -ST- 204) was first detected in building II in the third visit, and spread through this building until the end of the study. The answers to the questionnaire implied that lack of hygienic barriers in specific parts of the facilities and uncontrolled personnel flow were the critical factors for the spread of L. monocytogenes within and between buildings. Knowledge of the patterns of L. monocytogenes colonization can help a more rational design of new cheesemaking facilities, and improve the food safety within current facilities.