Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm-2) and H. perforatum (32 mg ml-1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml-1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.