The surface structures of probiotic bacteria are key molecules interacting with the microenvironment of the gastrointestinal tract, and some are being investigated as functional biomolecules. The representatives of these biomolecules are extracellular sugar polymers exopolysaccharides (EPS) and Sproteins, a group of surface proteins expressed by certain strains of Lactobacillus. Functional biomolecules of Lactobacillus spp. are intensively investigated and, according to recent research, are defined as postbiotics. Efficient extraction is crucial for the structural and functional analysis of target biomolecules. In this work, the optimization of EPS extraction conditions from selected Lactobacillus strains of the human milk microbiome was carried out. EPS was extracted from Limosilactobacillus fermentum MC1, and S-proteins from Levilactobacillus brevis strains MB1, MB2, MB13, MB20. Dialyzed samples of cell-bound EPS-b were isolated using three different agents: NaOH, phenol, and EDTA. The highest yield of YEPS=544.12 mg/L was determined in the EPS-b sample treated with NaOH. The maximum concentration of extracellular EPS (611.11 mg/L) (EPS-r) was experimentally determined in a sample extracted when trichloroacetic acid (TCA) was applied. SDS-PAGE of surface proteins established the presence of S-protein in all four strains of L. brevis. The concentration of extracted S-proteins was determined using the Bradford method, with the highest concentration (27.286 mg/µL) being determined in the sample of surface proteins of the strain L. brevis MB20.