Objective: Biofilms are surface-attached cell communities that play a role in the survival of bacteria. Helicobacter pylori, a gram-negative pathogen that colonizes the human gastric mucosa, forms biofilms, causing treatment failure and the risk of developing peptic ulcers, gastritis and gastric cancer in infected individuals. The aim of the study is to evaluate the biofilm formation abilities of H. pylori ATCC 26695 and three clinical strains in different culture media. Material and Method: Biofilm formation characteristics of H. pylori strains using different culture media were evaluated, and the crystal violet (CV) staining method (measured at OD 595 ) was used. Various media were used for incubating H. pylori strains: Brucella broth (BB), TSB with 10% FBS, BHI with 10% FBS, BB with 10% FBS, BB with 10% FBS + 0.25% glucose, and BB with 10% FBS + 1% glucose (incubated for 3 days). Additionally, BB with 1% FBS, BB with 10% FBS, and BB with 5% inactivated human serum were incubated for 2 and 4 days at 37°C under microaerophilic conditions. Results: It was observed that 5% inactived human serum was more effective in biofilm formation of H. pylori ATCC 26695 and three clinical strains. However, there was no biofilm production in the strains cultured with Brucella broth alone and that the strains cultured with TSB + 10% FBS could not form a strong biofilm compared to other media. Conclusion: Different culture media used for H. pylori ATCC 26695 and three clinical strains affect biofilm formation. It is thought that in vitro experiments to prevent biofilm formation may provide a solution to the prevention of H. pylori infection.