Antimicrobial Blue Light (aBL) can be used to control the growth of pathogens in several applicative fields, from sanitization of inert surfaces to human skin treatment and from industry to food. Though the mechanism of action is still unknown, it has been hypothesized that specific wavelengths can activate potential endogenous photosensitizers in microbial cytoplasm and/or envelope. In turn, this photooxidative stress could induce inactivation of macromolecules resulting in bacterial killing. In this work, we investigated the effect of radiometric parameters of light at 410 nm on Escherichia coli K-12 MG1655, a strain rather tolerant to blue light irradiation. Interestingly, by changing the radiometric parameters of aBL protocol, different rates of killing were observed. Irradiation at 100 J/cm² caused a variable antimicrobial effect depending on the irradiance values. We observed an “irradiance effect”: namely, at higher irradiance values, the inhibitory effect is reduced. On the other hand, at increasing fluences the bactericidal rate increases. In addition, the shift from continuous to pulsed light could enhance the antimicrobial activity of protocols using higher irradiance values. Taken together, these results underline the importance of defining radiometric parameters to ensure the efficacy of aBL treatments and emphasize the importance of further research into the aBL mechanism.