In the present study, a novel Pediococcus pentosaceus SP2 strain, recently isolated from kefir grains, was evaluated as a starter culture in sourdough bread making. The novel starter was applied in fresh, freeze-dried, and freeze-dried immobilized (on wheat bran) form. The type of culture (fresh, freeze-dried, immobilized cells) influenced the bread characteristics. Specifically, the application of freeze-dried immobilized cells led to higher total titratable acidity (TTA) values (9.81 mL NaOH N/10), and the produced bread presented higher resistance to mold and rope spoilage. Moreover, the produced sourdough breads were significantly better in terms of pH, TTA, organic acids content, and resistance to mold and rope spoilage, compared to breads made with a commercial, wild microbiota, sourdough. The organic acids content was also significantly higher than the commercial sourdough sample (2.93 g/kg lactic acid; 1.01 g/kg acetic acid). Determination of volatile compounds through solid-phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis and sensorial assessments indicated no significant differences between the tested sourdough breads.