Matsoni is a traditional Caucasian fermented milk product, characterised by a lactic acid bacteria (LAB) microbiota. Objectives of the present study were to isolate and identify the LAB of Matsoni samples collected from various Georgian areas and to study their technological characteristics. About 300 LAB from 71 samples of Matsoni were isolated and characterised. The species most representatives were Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus rhamnosus. This study highlighted a high degree of biodiversity and a specific geographical distribution of the dominant LAB present in artisanal Matsoni. Some strains showed technological features useful to design a multistrain starter culture for Matsoni.