Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation—owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane‐targeted azobenzene (Ziapin2) can be used to photo‐modulate the membrane potential in cells of the Gram‐positive bacterium Bacillus subtilis. It is found that upon exposure to blue–green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion‐channel‐deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid‐94 (IAA‐94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.