Bacterial antibiotic resistance is an alarming global issue that requires alternative antimicrobial methods to which there is no resistance. Antimicrobial photodynamic therapy (APDT) is a well-known method to combat this problem for many pathogens, especially Gram-positive bacteria and fungi. Hypericin and orange light APDT efficiently kill Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and the yeast Candida albicans. Although Gram-positive bacteria and many fungi are readily killed with APDT, Gram-negative bacteria are difficult to kill due to their different cell wall structures. Pseudomonas aeruginosa is one of the most important opportunistic, life-threatening Gram-negative pathogens. However, it cannot be killed successfully by hypericin and orange light APDT. P. aeruginosa is ampicillin resistant, but we hypothesized that ampicillin could still damage the cell wall, which can promote photosensitizer uptake into Gram-negative cells. Using hypericin and ampicillin cotreatment followed by orange light, a significant reduction (3.4 log) in P. aeruginosa PAO1 was achieved. P. aeruginosa PAO1 inactivation and gut permeability improvement by APDT were successfully shown in a Caenorhabditis elegans model.