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Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive <i>Bacillus subtilis</i> 168, the Gram-negative <i>Escherichia coli</i> DH5&#945; and <i>E. coli</i> strain NR698 which has a deficient outer membrane, simulating a Gram-negative &#8220;without&#8222; its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm², <i>B. subtilis</i> showed high sensitivity already at low chlorophyllin concentrations (&#8804;10⁵ cfu/mL: &#8804;0.1 mg/L, 10⁶&#8315;10⁸ cfu/mL: 0.5 mg/L), whereas <i>E. coli</i> DH5&#945; was less sensitive (&#8804;10⁵ cfu/mL: 2.5 mg/L, 10⁶ cfu/mL: 5 mg/L, 10⁷&#8315;10⁸ cfu/mL: ineffective at &#8804;25 mg/L chlorophyllin). <i>E. coli</i> NR698 was almost as sensitive as <i>B. subtilis</i> against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, <i>E. coli</i> NR698 and <i>B. subtilis</i> can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.