Traditional national cheeses are a source for the search for species and strains of lactic acid bacteria (LAB) which are not within the range of bacterial agents used in the dairy industry. The purpose of our work was isolation and identification of LAB from traditional Slovakian cheeses made from cow's milk. Forty samples of cheese were selected for our research, produced in the four regions of the Slovakia. LAB were cultured on de Man, Rogosa and Sharpe and cultivated for 48 hours at 37 °C in microaerophilic condition. The suspicious colonies from lactic acid bacteria were selected for further confirmation with MALDI-TOF. Selected colonies were cultured overnight on TSA agar anaerobically and used for identification. 166 cultures were isolated from the samples of cheese. Genus and species identification was completed using mass spectrometry method.  Based on the complex of LAB isolated were assigned to the following genera and species: Lactococcus spp., including L.  lactis and L.  garvieae; Lactobacillus spp.: L. curvatus, L. delbrueckii, L. fermentum, L. papacasei, L. paraplantarum, L. plantarum and L. sakei; Enterococcus spp.: E. durans, E. faecalis, E. faecium and E. italicus; Leuconostoc spp.: L. mesenteroides, Pediococcus spp.: P. pentosaceus and Streptococcus spp.: S. salivarius ssp. thermophilus.