This study investigated the survivals of two pathogens (Escherichia coli O157:H7 and Staphylococcus aureus) in different adhered forms on glass fiber filters (GFFs) at 43 and 68% relative humidity (RH). Efficacies of chemical sanitizers at reducing pathogenic biofilms on GFFs were also evaluated. Inoculated GFFs were incubated at 28 °C in TSB (type I), on TSA (type II), or on TSA under 100% RH (type III) to produce biofilms. When GFFs were incubated at 43 or 68% RH for 7 days, type III biofilms were less than 2 log10 CFU/filter reduction whereas type I and type II biofilms were 4-6 log10 CFU/filter reduction. Additionally, type III biofilms were highly resistant to sanitizing treatment compared than other biofilms (type I and II). Therefore, the method to produce biofilms used in this study could be used to produce highly resistance pathogenic biofilms in the laboratory for related experiments.