Gut microbiota is believed to play a crucial role in modulating obesity in humans, and probiotics affecting gut microbiota can alleviate some of the obesity-related health complications. The study was aimed to investigate changes in the composition of the gut microbiome in obese humans due to short-term (2 weeks) treatment of obese patients with a probiotic preparation containing Bifidobacterium longum. Faecal microbiome diversity was studied using the 16S amplicon sequencing by Illumina MiSeq. Bioinformatic analysis showed distribution across 14 phyla (with Firmicutes and Bacteroidetes dominating), 21 class, 125 genera and 973 OTUs. The probiotic treatment decreased relative abundance of Bacteroidetes (Prevotellaceae and Bacteroidaceae), while increasing that of Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae), and Firmicutes (Negativicutes: Veillonellaceae and Clostridia: Peptostreptococcaceae). The probiotic treatment decreased total blood sugar and increased patients’ assessment of their physical and mental health. Thus even the short-term Bifidobacterium-based probiotic treatment brought significant compositional changes in the 16S rRNA gene diversity in faecal bacterial assemblages by increasing beneficial and decreasing pathogenic or opportunistic bacteria; the related shifts in life quality assessment necessitate further research into the causal relationships involved.