In this study, two kinds of novel carbazole-based ethynylpyridine salts: 3,6-bis[2-(1-methylpyridinium)ethynyl]-9-pentyl-carbazole diiodide (BMEPC) and 3,6-bis[2-(1-methylpyridinium)ethynyl]-9-methyl-carbazole diiodide (BMEMC) have been employed as photosensitizers owing to their excellent antibacterial activity. These molecules possess symmetric A-π-D-π-A-type structures, which would bring in the unique optical properties. The inhibition zone measurement of a gradient concentration from 0 to 100 μM showed BMEPC and BMEMC photoinduced antibacterial activity against Escherichia coli. Diameters of zone of inhibition were up to 15 and 14 mm under laser irradiations. Under the exposure of the laser of 442 nm with a power density of 20 mW/cm2, the minimum inhibitory concentrations (MICs) of BMEPC on E. coli were between 3.5 and 6.9 μM and that of BMEMC were between 9.4 and 18.8 μM, respectively. In the dark experiments as a control, the MIC value is between 6.9 and 13.8 μM for BMEPC, whereas it is between 187.5 and 225.0 μM for BMEMC. By the comparison of the MIC values of BMEPC and BMEMC with laser irradiation and in dark, the laser-induced toxicity on bacteria is more evident, though both of the derivatives have dark toxicity. With the laser irradiation duration of 30 s and 10 min for BMEPC and BMEMC, respectively, the survival rate of E. coli approximates zero. An antibacterial mechanism has been proposed based on the electron paramagnetic resonance characterization, which indicates that a nitride radical is generated under laser irradiation. The carbazole-based ethynylpyridine photosensitizers would provide high potential for further applications in photodynamic therapy.