Disturbances in the composition of the gut microbiota, known as dysbiosis, may contribute to the development of obesity, as they negatively affect the efficiency of energy harvest and metabolic processes in the body. One promising therapeutic approach to improving intestinal homeostasis is the use of probiotic supplements. The present study aimed to comprehensively evaluate the effect of the probiotic strain Streptococcus salivarius on the gut microbiota of volunteers with obesity. Microbiome profiles of volunteers before and after administration of a probiotic supplement containing the bacterial species S. salivarius were obtained using high-throughput sequencing on the DNBSEQ-G50 platform. Alpha diversity analysis was performed using observed species richness and the Shannon diversity index. No statistically significant differences were detected. Beta diversity analysis showed clustering between the study groups; however, no statistically significant differences were found. Differential abundance analysis revealed statistically significant species-level differences between the microbiomes of participants before and after intake of the dietary supplement. After supplementation, volunteers showed a decrease in the abundance of the following species: Phocaeicola vulgatus (8.469% ± 2.884 vs. 3.460% ± 0.864, p = 0.013) and Yeguia hominis (0.017% ± 0.005 vs. 0.007% ± 0.004, p = 0.017). In contrast, the relative abundance of the taxon GGB2970 SGB3952 (Firmicutes) increased during probiotic supplementation compared with baseline values obtained before supplementation (0.043% ± 0.012 vs. 0.087% ± 0.009, p = 0.001). The decrease in the relative abundance of Phocaeicola vulgatus and Yeguia hominis, together with the increase in GGB2970 SGB3952, indicates a directed shift in the taxonomic profile of the gut microbiota. The direction of these changes may be characterized as potentially beneficial. However, the physiological significance of these shifts requires further investigation, including analysis of the functional potential of the microbiota and associations with clinical parameters.

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