Lactic acid bacteria have long been used in brewing to acidify mash or wort and in the production of traditional sour beer styles (Lambic, Gueuze) using spontaneous fermentation. This approach is time consuming (some sour beers are matured for three years to obtain the appropriate flavour), so many brewers choose a faster approach using mixed cultures (yeast and bacteria). In this study, the influence of the pitching sequence of bacteria and yeast on the fermentation process was evaluated (by rate of fermentation and acidification, attenuation degree and ethanol concentration). The trials were performed using three procedures: (i) the addition of bacteria followed by the yeast; (ii) the addition of the yeast followed by the bacteria and (iii) the simultaneous addition of yeast and bacteria. In each trial the following time intervals were applied: 24, 48 and 72 h. Weight loss of samples and drop in pH were measured daily, and ethanol content, real extract and lactic acid concentration measured at the end of the process. It was shown that the order of addition of yeast and bacteria determines the success of the lactic acid fermentation. The appropriate yield of lactic acid (ca. 6 g/L) and the drop in pH (<3.6) can be achieved by inoculating the bacteria prior to the inoculation of yeast. When yeast was pitched before the bacteria, the lactic acid content was ca. 2 g/L and pH was 3.9–4.2. © 2019 The Institute of Brewing & Distilling