The biofilm formation by foodborne pathogens is known to increase the problem related with surface disinfection procedure in the food processing environment and consequent transmission of these pathogens into the population. Messenger RNA has been increasingly used to understand the action and the consequences of disinfectants in the virulence on such biofilms. RNA quality is an important requirement for any RNA-based analysis since the quality can impair the mRNA quantification. Therefore, we evaluated five different RNA extraction kits using biofilms of the foodborne pathogens Listeria monocytogenes, Escherichia coli, and Salmonella enterica. The five kits yielded RNA with different quantities and qualities. While for E. coli the variability of RNA quality did not affect the quantification of mRNA, the same was not true for L. monocytogenes or S. enterica. Therefore, our results indicate that not all kits are suitable for RNA extraction from bacterial biofilms, and thus, the selection of RNA extraction kit is crucial to obtain accurate and meaningful mRNA quantification.