Efficient inactivation of Escherichia coli (E. coli) under visible (532 nm) pulsed light irradiation was achieved by fusion of a visible light-absorbing dye with E. coli. Inactivation experiments showed that 3-log inactivation of E. coli was obtained within 20 min under a 50 kJ/cm2 dose. This treatment time and dose magnitude were 10 times faster and 100 times lower, respectively, than the values previously obtained by using a visible femtosecond laser. The mechanism of bacterial death was modeled based on a transient photothermal evaporation effect, where a quantitative evaluation of the temperature increase was given based on the heat transfer equation. As a result of this theoretical analysis, the maximum temperature of the bacteria was correlated with the absorption ratio, pulse energy, and surface-to-volume ratio. An increase in the surface-to-volume ratio with the decreasing size of organic structures leads to the possibility of efficient inactivation of viruses and bacteria under low-dose and non-harmful-visible pulsed light irradiation. Hence, this method can be applied in many fields, such as the instantaneous inactivation of pathogenic viruses and bacteria in a safe and simple manner without damaging large organic structures.