This study illustrates the effectiveness of slightly acidic hypochlorous water (SAHW) in comparison with sodium hypochlorite (NaOCl) in reducing biomass and viable cells in biofilms established by the dual species, Listeria monocytogenes and Escherichia coli, on a microtiter plate and stainless-steel coupon. The SAHW and NaOCl treatments exhibited significant efficacy against biofilms (p < 0.05) on both surfaces. Additionally, compared with NaOCl treatment, SAHW treatment significantly reduced biofilm formation (p < 0.05). With its high antibiofilm activity, SAHW not only reduced the biomass of biofilms, but also significantly decreased viable biofilm cells to 5 log CFU/mL or ≤1 log CFU/cm2 on microtiter plates and stainless-steel surfaces, respectively. These results indicate that SAHW is a potential candidate for disinfectants against biofilms on various food contact surfaces.