Abstract <p>This study evaluates the combined application of MALDI-TOF MS and Fourier transform infrared (FT-IR) spectroscopy for the purpose of species identification and strain-level differentiation of lactic acid bacteria isolated from commercial yoghurts. MALDI-TOF MS provided rapid species-level identification, while FT-IR detected metabolic variations by analysing key cellular components such as membrane fatty acids (3000–2800&#xa0;cm⁻¹), protein amide bands (1800–1500&#xa0;cm⁻¹), and polysaccharides (1200–900&#xa0;cm⁻¹). These spectral features reflected phenotypic differences among strains linked to antibiotic resistance profiles. Disc diffusion antibiotic susceptibility testing revealed significant variability among isolates, and a strong correlation was observed between FT-IR-derived linear discriminant analysis (LDA) clusters and resistance to oxacillin, clindamycin, and tetracycline (Chi² test, <i>p</i> &lt; 0.05). This demonstrates FT-IR as a rapid, non-invasive tool for the early detection of resistant strains, facilitating real-time monitoring of bacterial adaptation during fermentation. The findings of this study provide support for integrating FT-IR and MALDI-TOF MS into industrial quality control, aiding in strain selection and enhancing food safety.</p> Key points <p>• <i>FT-IR enables rapid phenotypic typing of lactic acid bacteria strains.</i></p> <p>• <i>Spectral profiles correlate with antibiotic resistance.</i></p> <p>• <i>MALDI-TOF MS and FT-IR offer a robust, complementary identification method.</i></p>