BackgroundInfection withHelicobacter pylorias the cause of gastric cancer is a global public health concern. In addition to protecting germs from antibiotics, biofilms reduce the efficacy ofH. pylorieradication therapy. The nucleotide polymorphisms (SNPs) related with the biofilm forming phenotype ofHelicobacter pyloriwere studied.ResultsFifty-sixH. pyloriisolate from Bangladeshi patients were included in this cross-sectional study. Crystal violet assay was used to quantify biofilm amount, and the strains were classified into high- and low-biofilm formers As a result, strains were classified as 19.6% high- and 81.4% low-biofilm formers. These phenotypes were not related to specific clades in the phylogenetic analysis. The accessories genes associated with biofilm from whole-genome sequences were extracted and analysed, and SNPs among the previously reported biofilm-related genes were analysed. Biofilm formation was significantly associated with SNPs ofalpA, alpB, cagE, cgt, csd4, csd5, futB, gluP, homD, andmurF(P < 0.05). Among the SNPs reported inalpB, strains encoding the N156K, G160S, and A223V mutations were high-biofilm formers.ConclusionsThis study revealed the potential role of SNPs in biofilm formation and proposed a method to detect mutation in biofilm from whole-genome sequences.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02889-8.