This study reports the characterization of Pediococcus acidilactici strain K1–UzRSMMT-396, a biosurfactant (BS)-producing lactic acid bacterium isolated from the traditional fermented milk product qurt. Primary screening of lactic acid bacteria isolates revealed a strain with pronounced surface-active properties, indicating its potential as a BS producer. Species identification was confirmed using morphological, biochemical, and molecular approaches, including MALDI-TOF MS and full-length 16S rRNA gene sequencing. The strain exhibited susceptibility to most of the tested antibiotics, including chloramphenicol, erythromycin, amikacin, cefotaxime, vancomycin, streptomycin, and amoxicillin/clavulanate, while showing moderate resistance to ciprofloxacin. P. acidilactici K1–UzRSMMT-396 demonstrated tolerance to simulated gastrointestinal conditions. After 90 min of exposure to simulated gastric juice (pH 2.0), viable cell counts remained at 5.1 ± 0.04 log CFU/mL, indicating the survival of a substantial fraction of the initial population. In the presence of bile salts, the strain maintained high viability, suggesting tolerance to conditions mimicking the small intestinal environment. In addition, the strain exhibited osmotolerance, retaining viability at elevated NaCl concentrations in MRS medium. Optimization of the cultivation medium showed that reducing cheese whey concentration from 50%-30% and halving the amounts of glucose, yeast autolysate, and ammonium citrate enhanced BS production. The emulsification index after 24 hrs of cultivation (EI24) reached 70%-74%, and the maximum BS yield was 2.704 g/L. The isolated biosurfactant exhibited pronounced antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, and effectively disrupted P. aeruginosa biofilms, resulting in a 59%-67% reduction in biofilm biomass depending on the test strain. These findings indicate that P. acidilactici strain K1–UzRSMMT-396 and its biosurfactant have strong potential for application in the food and pharmaceutical industries as safe antimicrobial and anti-adhesive agents.