This study was conducted to compare the effect of different acidification methods using Lactobacillus amylovorus FST2.11 as a starter culture on the microbiological, technological, and qualitative attributes of sour beers. Biological souring was performed in the mash after mashing, in the preboiling wort, or in the postboiling wort. This lactic acid bacterium strain was selected for its high sensitivity to hops, good growth at moderate levels of alcohol, and fast acidification in wort. Alcoholic fermentation was carried out using a commercial Saccharomyces cerevisiae (Safale US-05) strain. Desired acidification of the unhopped substrates (approximately 5–6 g/L of lactic acid) was achieved within 18 h of lactic fermentation. The lactic culture consumed maltose preferentially over monosaccharides, and uptake of free amino nitrogen was limited (8.0–12.6 mg/L). Yeast growth in soured substrates was delayed by 2–4 days compared with the unacidified control, but comparable end attenuations were achieved among all treatments. Among the soured beers, the lowest levels of off-flavors were found in preboil wort souring trials, whereas cofermented beers led to opalescent beers containing high levels of total diacetyl and acetoin. The low pH and the high level of staling compounds such as acetaldehyde suggest that sour beers suffered from low oxidative stability. Final beer qualities varied considerably depending on the souring method applied. Souring of wort by L. amylovorus FST2.11 before boiling was found to produce bright, tart beer with minor organoleptic failures while limiting the risk of bacterial cross-contamination within the brewing facility. The renewed interest that sour brewing has received among brewing communities worldwide calls for a better understanding of the advantages and disadvantages of different souring methods. The results from this study could help brewers to choose the most suitable approach according to equipment capabilities and sensorial likeness.