Salmonella is a common foodborne infection. Many serovars belonging to Salmonella enterica subsp. enterica are present in the gut of various animal species. They can cause infection in human infants via breast milk or cross-contamination with powdered milk. In the present study, Salmonella BO was isolated from human milk in accordance with ISO 6579-1:2017 standards and sequenced using whole-genome sequencing (WGS), followed by serosequencing and genotyping. The results also allowed its pathogenicity to be predicted. The WGS results were compared with the bacterial phenotype. The isolated strain was found to be Salmonella enterica subsp. enterica serovar Typhimurium 4:i:1,2_69M (S. Typhimurium 69M); it showed a very close similarity to S. enterica subsp. enterica serovar Typhimurium LT2. Bioinformatics sequence analysis detected eleven SPIs (SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14, C63PI, CS54_island). Significant changes in gene sequences were noted, causing frameshift mutations in yeiG, rfbP, fumA, yeaL, ybeU (insertion) and lpfD, avrA, ratB, yacH (deletion). The sequences of several proteins were significantly different from those coded in the reference genome; their three-dimensional structure was predicted and compared with reference proteins. Our findings indicate the presence of a number of antimicrobial resistance genes that do not directly imply an antibiotic resistance phenotype.