The majority of chronic infections are caused by biofilms, which have higher levels of antibiotic resistance than planktonic growth. Violet-blue 405 nm light has recently emerged as a novel bactericide, but limited studies have been conducted on its effectiveness against biofilms. We found that in response to 405 nm light both Pseudomonas aeruginosa and Bacillus subtilis biofilms exhibited cell dispersal and membrane potential hyperpolarisations. The response to 405 nm light depended on the stage of biofilm growth. The use of reactive oxygen species scavengers reduced membrane hyperpolarisation and biofilm dispersal in response to 405 nm light. This is the first time that membrane potential hyperpolarisations have been linked with photooxidative stress in bacteria and with biofilm dispersal. These results provide a new insight into the role of membrane potentials in the bacterial stress response and could be used in the development of 405 nm light based biofilm treatments.