Evolving antibiotic resistance of bacteria is a prevailing global challenge in health care and requires the development of safe and efficient alternatives to classic antibiotics. Photodynamic Inactivation (PDI) has proven to be a promising alternative for treatment of a broad range of microorganisms. Photodynamic Inactivation uses photoactive molecules that generate reactive oxygen species (ROS) upon illumination and in the presence of oxygen, which immediately kill pathogenic target organisms. Relevant photoactive properties are provided by berberine. Originally extracted from Barberry (Berberis vulgaris), it is a natural compound widely used in Traditional Chinese Medicine for its antimicrobial and anti-inflammatory effects. With this study, we demonstrated the potential of berberine chloride hydrate (Ber) as a photosensitizer for PDI of important human pathogens, Gram(+) Staphylococcus capitis subsp. capitis, Gram(+) Staphylococcus aureus, and Gram(−) Escherichia coli. In vitro experiments on planktonic and biofilm cultures were conducted focusing on Ber activated with visible light in the blue wavelength range. The number of planktonic S. capitis cells was reduced by 7 log10 steps using 100 µM Ber (5 min incubation, illumination with 435 nm LED array, radiant exposure 25 J/cm2). For an antibacterial effect of 4 log10 steps, static S. capitis biofilms required 1 mM Ber, a drug-to-light interval of 60 min, and illumination with 100 J/cm2. Almost all planktonic cells of Staphylococcus aureus could be photokilled using 100 µM Ber (drug-to-light interval of 30 min, radiant exposure 25 J/cm2). Biofilms of S. aureus could be phototreated (3 log10 steps inactivation) when using 1 mM Ber incubated for 5 min and photoactivated with 100 J/cm2. The study is highlighted by the proof that PDI treatment using Ber showed an antibacterial effect on Gram(−) E. coli. Planktonic cells could be reduced by 3 log10 steps with 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). With 5 mM ethylenediamine tetraacetic acid disodium salt dihydrate (Na2EDTA) or 1.2% polyaspartic acid (PASA) in addition, a relative inactivation of 4 log10 steps and 7 log10 steps, respectively, was detectable. Furthermore, we showed that an antibacterial effect of 3.4 log10 towards E. coli biofilms was given when using 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). These results underscore the significance of PDI-treatment with Ber as a natural compound in combination with blue light as valuable antimicrobial application.