Objective In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. Materials and methods Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms. Results The number of colony-forming units in the R. officinalis–treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64–2.78 and 2.41–3.23 for aerobic and anaerobic bacteria, respectively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylobacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis–treated biofilms. Live/dead staining confirmed that the R. officinalis–treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms. Conclusions The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. Clinical relevance The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances.