Preliminarily, 146 strains of yeasts were screened for the antifungal activity toward the indicator Penicillium roqueforti DPPMAF1. The strain Meyerozyma guilliermondii LCF1353 was selected and used for dough fermentation. The water/salt soluble extract of the dough was analyzed by HPLC and GC/MS-SPME. The synthesis of the extracellular cell wall-degrading enzyme β-1,3-glucanase and ethyl-acetate was shown. The effect on conidia germination mainly suggested a fungistatic activity. M. guilliermondii LCF1353 was used as starter for dough fermentation in combination with Wickeramomyces anomalus 1695 and Lactobacillus plantarum 1A7, which were previously selected for antifungal activity. The growth of the strains was monitored by plate count and molecular techniques, and competitive or antagonistic interactions among them were excluded. Bread started with the combination of M. guilliermondii LCF1353, W. anomalus LCF1695 and L. plantarum 1A7 showed a more prolonged shelf life compared to the other breads. Fungal growth was delayed at least until 14 days of storage, under conditions of high artificial inoculum. The bread manufactured with the above combination showed good chemical and textural characteristics and, as shown by sensory analysis, it was appreciated for elasticity, color and taste. ► 146 strains of yeasts were screened for the antifungal activity. ► Meyerozyma guilliermondii LCF1353 was selected for antifungal activity. ► Antifungal activity was characterized by in vitro assays. ► The synthesis of ethyl-acetate, ethanol and of a cell wall-degrading enzyme was shown. ► Breads fermented with the selected yeast were tested during storage.