DescriptionBiofilms were defined as the structural phenotype of microbial communities enclosed in the self-produced polymeric matrix mainly composed of extracellular polysaccharides (EPS). S.epidermidis is associated with chronic diseases involving implant medical devices due to its strong ability to form biofilms and they are difficult to eradicate. Rifampin, one of the most active antibiotics used to treat biofilm-associated infections, is a bacterial RNA polymerase (RNAP) inhibitor; however strong bacterial resistance to this drug leads to the need to find more active compounds acting at the same target: bacterial RNAP. In this study, I test whether other bacterial RNAP inhibitors are also active against S.epidermidis biofilms by determining their Minimum Biofilm Eradication Concentration (MBEC) as compared to those of antibiotics functioning through target other than bacterial RNAP (reference antibiotics). Two biofilm assays were used: MBEC P&G assay using Calgary Biofilm device (peg assay) and glass biofilm assay. In the peg assay, biofilm density of 6 log10 of cfu/peg was obtained and used for biofilm susceptibility testing. In addition to Rifampin, three novel synthesized bacterial RNAP inhibitors were found to be more effective in completely eradicate S.epidermidis biofilms in vitro: 3RHTK27, 3RHTK44 and OMTK13 with MBEC value of 25µg/ml, 12.5µg/ml and 50µg/ml, respectively. None of the other antibiotics were able to eradicate S.epidermidis biofilms (MBEC > 400µg/ml). These three RNAP inhibitors were also found to be effective in glass biofilm assay having MBEC values of 6.25µg/ml, 3.125µg/ml and 25µg/ml, respectively. These three compounds are interesting candidates for further development as antibacterial and anti-biofilm agents. Not all RNAP inhibitors which are active against planktonic cells are active against S.epidermidis biofilms. Nevertheless, seeing the same pattern of result from two different assays that reference antibiotics tested were not capable of eradicating biofilms while three out of eleven bacterial RNAP inhibitors tested were active is encouraging and suggesting that RNAP makes a good target for finding drugs to treat biofilm-related diseases.