Introduction: Biocides are commonly used for disinfection in a variety of contexts. They are generally used to avoid infection by controlling biofilm on medical equipment. However, the literature lacks information on the effect of biocide on efflux pump gene expression. Objective: To determine the influence of biocide on biofilm development and efflux pump acrA and ramA gene expression. Methodology: The microtiter plate method was used to identify biofilm development in 80 isolates of K. pneumoniae. The minimal inhibitory concentrations (MIC) of three biocides (quaternary ammonium compound (QAC), chlorohexidine digluconate, and chloroxylenol) were estimated. The effect of QAC on the intensity and viability of biofilms was investigated as well. Its influence on biofilm was evaluated using scanning electron microscopy. For each isolate, gene expression of the acrA and ramA was evaluated before and after treatment with the biocide (at ½MIC). Results: The study revealed that 60% of isolates formed moderate biofilms. Biofilms of all isolates were inhibited by QAC at high concentrations. Moreover, QAC at ½MIC downregulated the gene expression of the efflux pump gene (acrA) and its regulator (ramA). Conclusion: Biofilms of all isolates were prevented by QAC at high doses. Furthermore, QAC at ½MIC decreased the expression of the efflux pump gene (acrA) and its regulator (ramA).