The MALDI-TOF MS technique was used by our team to identify microorganisms present in milk samples. The same strains were identified using two instruments, Microflex LT/SH (Bruker, Germany) and EXS2600 (Zybio Inc., China). The results obtained indicated differences in the identification of some of the bacteria isolates. Therefore, as a complementary technique, we decided to use the innovative NALDI-TOF MS technique for the analysis. It is an example of a matrix-free approach that enables the analysis of low-molecular compounds such as lipids. Within NALDI-TOF-MS organic matrices are replaced with nanoparticles or nanostructures deposited as a thin layer on an appropriate substrate. They are responsible for the enrichment of analytes and provide efficient desorption and ionization based on surface plas-mon resonance. NALDI-TOF MS was used to analyse bacterial lipid extracts on a specially prepared plate with deposited silver nanoparticles (AgNPs). The results indicate the potential use of NALDI-TOF MS in the identification and differentiation of closely related microorganisms.