Sourdough is a very interesting natural preservation system to prolong mould free shelf-life of bread. Numerous studies have reported that the antifungal activity of sourdough is mainly correlated with the presence of lactic (LA) and acetic acid (AA), but very few information is available on the effect of undissociated acid concentrations in the aqueous phase of bread (C; mmole/L). This study was conducted to provide additional information about the mode of action of the acids in sourdough bread, enabling a better shelf-life prediction. This study was divided into two parts. In part 1, three industrial biological sourdoughs were characterized (dough yield, pH, a, fermentation quotient, microbiota). During 7 weeks, a shelf-life test with natural flora was conducted with daily checks of visible mould growth (21 °C). In part 2, the effect of the acids present in the antifungal active sourdough breads was validated in chemically acidified wheat breads. Complete growth inhibition was observed in full-baked sourdough bread (30 g/100 g dough) containing Lactobacillus sanfranciscensis and Saccharomyces cerevisiae as dominant sourdough micro-organisms, whereas in control bread the shelf-life was limited to 4.4-9.2 days. These full-baked sourdough breads contained 36 mmole undissociated LA/L and 220 mmole undissociated AA/L. The data were used to make General Linear Regression models for shelf-life prediction and resulted in a fit of R = 0.79 when expressing the shelf-life in function of C and C. In acidified breads, the role of lactic acid was not significant and only impacted shelf-life indirectly through acidification. No difference between antifungal activity of sourdough breads and chemically acidified bread with comparable C concentrations was observed. Shelf-life increased when 150-200 mmole undissociated AA/L aqueous phase in bread was present. To conclude, this study showed the importance of the undissociated acid fraction of acetic acid in relation to bread shelf-life, together with bread pH and moisture content.