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Polyethylenimines (PEIs), a group of polycationic molecules, are known to impair the outer membrane of Gram-negative bacteria and exhibit antimicrobial activity. The outer membrane of Gram-negative strains hinders the uptake of photosensitizer chlorophyllin. In this study, we report chlorophyllin and branched PEI combinations’ activity against Escherichia coli strains DH5α and RB791, Salmonella enterica sv. Typhimurium LT2, and Bacillus subtilis 168. The minimal bactericidal concentration (MBC) was determined by plating cells treated with different concentrations of PEI and chlorophyllin on agar and monitoring their growth after 24 h. All tested combinations of PEI and chlorophyllin were lethal for S. enterica after 240 min of incubation in light, whereas PEI alone (−1) was ineffective. In the darkness, complete inhibition was noted with a combination of ≥2.5 µg mL−1 chlorophyllin and 50 µg mL−1 PEI. If applied alone, PEI alone of ≥800 µg mL−1 of PEI was required to completely inactivate E. coli DH5α cells in light, whereas with ≥5 µg mL−1 chlorophyllin, only ≥100 µg mL−1 PEI was needed. No effect was detected in darkness with PEI alone. However, 1600 µg mL−1 PEI in combination with 2.5 µg mL−1 resulted in complete inactivation after 4 h dark incubation. PEI alone did not inhibit E. coli strain RB791, while cells were inactivated when treated with 10 µg mL−1 chlorophyllin in combination with ≥100 µg mL−1 (in light) or ≥800 µg mL−1 PEI (in darkness). Under illumination, B. subtilis was inactivated at all tested concentrations. In the darkness, 1 µg mL−1 chlorophyllin and 12.5 µg mL−1 PEI were lethal for B. subtilis. Overall, PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin.