Objective: Bacterial 16S ribosomal DNA (rDNA) sequences are widely used to determine taxonomic and phylogenetic classification of human bacteriome. The selection of the hypervariable sub-regions to be analyzed may affect the analysis results, which may lead to incorrect interpretation of data. Selection of appropriate validated methodology in next-generation sequencing using 16S rRNA is, therefore, crucial. This work aimed to compare results obtained from 16S rRNA sequencing analysis of V1/V2 vs. V3/V4 region. Methods: Six patients with BE/EAC were included in this study. DNA from six oral swabs and twelve esophageal biopsies from Barrett's mucosa/adenocarcinoma and from healthy sites were isolated using the AllPrep DNA/RNA Kit (Qiagen). The V1/V2 region of the 16S rRNA gene was amplified using PCR and modified primers 68Fmod and 338R. The metagenomic library was prepared using the MiniSeq® High Output Kit (2 x 150 paired end sequencing) and deeply sequenced on an Illumina Miniseq 150bp instrument.    The V3/V4 16S rRNA sequencing analysis was performed on the same samples, which were, in addition, spiked by a mock community (Allobacillus and Imtechella, microbial concentration 104; ZymoBIOMICS Spike-in Control I, High Microbial Load); six negative controls (NC, DNA-free water) were added to the analysis. The V3/V4 region of the gene for 16S rRNA was amplified using Illumina sequencing primers. The metagenomic library was prepared using the Nextera DNA Flex Library Prep Kit V3 and deeply sequenced on an Illumina MiSeq instrument.   Results: The number of reads per sample was generally higher in samples sequenced using the approach focusing on V3/V4, which led to the higher resolving power than the V1/V2 method. In other words, more taxa were identified by V3/V4 16S rRNA sequencing than by V1/V2 16S rRNA sequencing. The MDS Canberra distance analysis showed a regular shift between V1/V2 and V3/V4 analyses, which indicates that both methods yield a similar trend with a systematic difference rather than random error. Conclusion: In studied matrices, the relative abundance of taxa using 16S rRNA sequencing of the V3/V4 region were more consistent with literature (i.e., detected a high abundance of Streptococcus) than the V1/V2 method. We, therefore, conclude that the V3/V4 approach with internal standard is more suitable for analysis of oral and esophageal bacteriome than the V1/V2 method.