This study aimed at assessing the effect of tap water on the: (i) lactic acid bacteria (LAB) population of a traditional and mature sourdough; and (ii) establishment of LAB community during sourdough preparation. Ten tap water, collected from Italian regions characterized by cultural heritage in leavened baked goods, were used as ingredient for propagating or preparing firm (type I) sourdoughs. The same type and batch of flour, recipe, fermentation temperature and time were used for propagation/preparation, being water the only variable parameter. During nine days of propagation of a traditional and mature Apulian sourdough, LAB cell density did not differ, and the LAB species/strain composition hardly changed, regardless of the water. When the different tap water were used for preparing the corresponding sourdoughs, the values of pH became lower than 4.5 after two to four fermentations. The type of water affected the assembly of the LAB biome. As shown by Principal Components Analysis, LAB population in the sourdoughs and chemical and microbiological features of water used for their preparation partly overlapped. Several correlations were found between sourdough microbiota and water features. These data open the way to future researches about the use of various types of water in bakery industry.