The aim of this work was to study the fungal colonization of a new winery over time, specifically for Saccharomyces cerevisiae. Therefore, we analyzed the flora present before the arrival of the first harvest on the floor, the walls and the equipment of this new winery by Illumina MiSeq. The genus Saccharomyces (≤0.3%) was detected on floor and equipment but the presence of S. cerevisiae species was not reported. Wild S. cerevisiae strains were isolated from a ‘Pied de Cuve’ used during the first vintage to ensure the alcoholic fermentation (AF). Among 25 isolates belonging to this species, 17 different strains were identified highlighting a great intraspecific diversity. S. cerevisiae strains were also isolated from different vats throughout the spontaneous fermentations during the first vintage. The following year, some of these strains were isolated again during AF. Some of them (four) were found in the winery equipment before the arrival of the third harvest suggesting a potential colonization by these strains. To better understand what promotes the yeast colonization of the winery’s environment, the ability to form a biofilm on solid surfaces for eight colonizing or non-colonizing strains was studied. This capacity, different according to the strains, could partly explain the colonization observed for certain strains