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Introduction: Dental biofilm is the main reason for several oral diseases such as caries and periodontitis. Therefore, there is a high demand for preventative strategies such as the use of chemical agents for the control of dental biofilm. As a consequence, it is always a priority interest and an important research area in the field of dentistry to evaluate and compare the effectivity of different antibacterial agents and to investigate the retention, stability, and efficacy of different treatment regimens. Objective: This in situ study aims to determine the retention of chlorhexidine in different oral locations after the application of various chlorhexidine regimens. Furthermore, it seeks to investigate and compare the anti-biofilm efficacy of chlorhexidine (CHX) and octenidine (OCT) mouth rinses. Material and Methods: Five volunteers were recruited for this work. In the first retention experiment, the samples were taken with micro-brushes from the interdental area, buccal dental pellicle, anterior labial and posterior buccal mucosa, as well as saliva at five-time points within 24 h after mouth rinsing with 10 ml of 0.2% CHX for 30 s. In the second retention experiment, the volunteers used different CHX treatment regimens from mouth rinses, dental spray, or toothpaste. After application, 2 µl samples were taken from the saliva and buccal mucosa pellicle in addition to the dental pellicle sample formed on a standardized enamel surface. The samples were taken at six-time points within 12 h. Retention of CHX in the aforementioned experiments was measured using Matrix-assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. In the second part of this work, biofilms were formed in the anti-biofilm experiment in situ on bovine enamel surfaces fixed to individual acrylic splints. For biofilm formation analysis, the subjects intraorally exposed the splint for 48 h. Mouth rinses were done using 10 ml of 0.1% CHX, 0.2% CHX, 0.1% OCT, or water as control. Each rinsing was performed every 12 h for 30 s. Biofilm colonization and bacterial vitality were investigated at 24 and 48 h. For analysis of biofilm disruption activity, the evaluation of 48 h-mature biofilms was performed before and directly after the first CHX rinse in both concentrations or OCT rinse and repeated 24 h later for the 72 h-mature biofilms. The coverage, vitality, and ultrastructure assessments of biofilm samples were performed by fluorescence microscopy and transmission electron microscopy. Results: A significant reduction of the intraoral CHX concentration was detected during the first 6 h. Thereafter, CHX retained in the oral cavity at low concentrations of micrograms per millilitre levels for 12 h after application. CHX was even detected 24 h after oral rinsing. CHX retention in the oral mucosa and dental pellicle was significantly higher than the retention in saliva. The retention of CHX after mouth rinsing or spray application was higher compared to the retention after the use of toothpaste. Rinses with 0.1% CHX, 0.2% CHX, or 0.1% OCT caused a significant reduction in the biofilm formation on enamel surfaces. Bacterial colonization and vitality were both clearly reduced. Moreover, a remarkable biofilm disruption was observed due to the application of CHX or OCT rinses. Interestingly, only one application of CHX or OCT to a 48-h mature biofilm induced biofilm ultrastructure modification and significantly reduced the biofilm thickness and microbial vitality. Conclusion: It was found in the present work that the MALDI-TOF technique provides excellent quantification limits for the CHX determination. It allows the detection of CHX without the need for time-consuming sample preparation. The obtained results indicate that dental pellicle and oral mucosa are the essential reservoirs of CHX in the oral cavity. With time, the CHX is desorbed from the oral surfaces toward saliva to produce a persistent bacteriostatic effect in the whole oral cavity. Moreover, the results show that there are significant differences in CHX retention after the application of different CHX regimens; mouth rinses, dental spray, and toothpaste. It also was concluded in this work that the mouth rinses based on CHX in both concentrations 0.1% and 0.2% as well as 0.1% OCT have a significant effect on reducing biofilm formation confirmed through the reduction of the oral bacterial colonization and biofilm vitality on native enamel specimens. Additionally, this study demonstrates an important biofilm disruption effect of the CHX and OCT rinsing, since they were effective to dislodge pre-existing biofilms, even in the total absence of mechanical biofilm management. However, 0.1% OCT showed significantly better results in anti-biofilm activity in comparison with CHX at the same concentration.