Multidrug-resistant bacteria represent a significant challenge in the treatment of bacterial infections, often leading to therapeutic failures. This issue underlines the need to develop strategies that improve the efficacy of conventional antibiotic therapies. In this study, we aimed to assess whether a plant-derived compound, α-mangostin, and photodynamic therapy (PDT) could enhance the antibacterial activity of ciprofloxacin against uropathogenic strains of and . Using nanopore sequencing technology, we confirmed that the clinical strains tested were classified as multidrug-resistant. Digital holotomography (DHT) was used to examine α-mangostin-induced changes in the bacterial cells' penetration by a photosensitizer. A scanning confocal fluorescence microscope was used to visualize photosensitizer penetration into bacterial cells and validate DHT results. A synergistic effect between α-mangostin and ciprofloxacin was observed exclusively in strains, while no enhancement of ciprofloxacin's antibacterial activity was detected in strains when combined with α-mangostin. Notably, photodynamic therapy significantly potentiated the antibacterial effects of ciprofloxacin and its combination with α-mangostin compared to untreated controls. In addition, morphological changes were observed in bacterial cells exposed to these antimicrobials. In conclusion, our findings suggest that α-mangostin and PDT may serve as valuable adjuncts to ciprofloxacin, improving the eradication of uropathogens.