Objective To investigate the features and mechanism of action of intestinal flora in patients with nonalcoholic fatty liver disease （NAFLD） and Helicobacter pylori （HP） infection by comparing the changes in intestinal flora between the healthy population，the patients with HP infection，the patients with NAFLD，and the patients with NAFLD and HP infection. Methods This study was conducted among the 19 patients with NAFLD （NAFLD group），19 patients with HP infection （HP group），and 19 patients with NAFLD and HP infection （NAFLD+HP group） who were admitted to The Second Affiliated Hospital of Henan University of Science and Technology from March 1，2023 to April 30，2024，and 20 individuals undergoing physical examination were enrolled as control group. Fecal samples were collected，total DNA was extracted for PCR amplification，and 16S rDNA sequencing was performed to compare the features of intestinal flora between the four groups. An analysis of variance was used for comparison of continuous data between multiple groups，and the chi-square test was used for comparison of categorical data between multiple groups. The Mann-Whitney U test or the Kruskal-Wallis H test was used for comparison of the species in intestinal flora. Results The NAFLD+HP group showed a tendency of reduction in flora abundance compared with the other three groups. There was a significant difference in flora distribution between the NAFLD+HP group and the NAFLD group and between the NAFLD group and the control group （P<0.05）. At the phylum level，the top three species in the NAFLD+HP group were Firmicutes （59.94%），Proteobacteria （17.00%），and Actinobacteria （14.75%），with an increase in the proportion of Proteobacteria and a reduction in the proportion of Actinobacteria compared with the other three groups. At the genus level，the top five dominant bacteria in the NAFLD+HP group were Bifidobacterium，Streptococcus，Escherichia-Shigella，Agathobacter，and Ruminococcus gnavus_group. Compared with the NAFLD group，the NAFLD+HP group had increases in the abundance of Streptococcus，Veillonella，and Rothia and reductions in the abundance of Dialister and Ruminococcus toraues_group. Compared with the HP group，the NAFLD+HP group had reductions in the abundance of Collinsella，Subdoligranulum，Catenibacterium，and Porphyromonas and increases in the abundance of Citrobacter and Olsenella （all P<0.05）. Conclusion Patients with NAFLD and HP infection have changed in intestinal flora. These flora may be the intestinal microecological factors for HP infection in promoting the development and progression of NAFLD.