The histological changes in the gastric epithelium are crucial in the progression from premalignant to neoplastic lesions. TLR4, TLR5 and TLR9 have been localized in the gastric epithelium and studied separately using conventional histological techniques without a focus on the protein or cell interactions within the microenvironment. Therefore, we developed a multiplex immunohistochemistry/immunofluorescence (mIHC/IF) technology for the simultaneous detection of TLR4, TLR5 and TLR9 on a single tissue section of human gastric biopsies from 10 paired cases collected in two independent visits, and its correlation with the OLGA/OLGIM scoring and H. pylori status after eradication. The results confirmed that mIHC/IF is useful for simultaneously interrogating six biomarkers and demonstrated that TLR4 and TLR9 are significantly associated with H. pylori infection. However, only TLR9 is positively related to the presence of intestinal metaplasia. TLR5 was mainly present in goblet cells (TFF3+) but did not show any significant association with H. pylori or the presence of intestinal metaplasia. Our results suggest that a more comprehensive strategy to interrogate the tissue microenvironment in premalignant lesions may improve the interpretation of the earned risk of gastric cancer in patients with chronic gastritis and evidence of failure in H. pylori eradication.