The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli ( EcoliBL21Lh β-gal) and Lactobacillus plantarum ( Lp609Lh β-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus , together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve , was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ∼90% of all GOS components. GOS mixtures obtained with β-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).