Aim: To evaluate in vitro the antibacterial efficacy of Matricaria recutita (chamomile) essential oil at 50 and 75% against Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611 at 24 and 48 hours. Material and methods: The sample consisted of 80 discs and Mueller–Hinton Agar, the medium chosen for the culture. To determine the bacterial sensitivity, discs were placed in each Petri dish with concentrations of essential oil at 50 and 75%, distilled water and 0.12% chlorhexidine. Subsequently, the inhibition halos were measured in millimeters at 24 and 48 hours after culture, with the Kirby–Bauer disk diffusion method. Results: In groups treated with Porphyromonas gingivalis, measurements at 24 and 48 hours yielded 22.14 ± 2.61 and 22.63 ± 2.67 mm for 0.12% chlorhexidine, 18.90 ± 0.41 and 19.22 ± 0.54 mm for 75% essential oil, and 15.55 ± 0.45 and 15.77 ± 0.46 mm for 50% essential oil, respectively. No statistically significant differences were observed among the groups (p > 0.05). Conclusion: No significant differences were found between the antibacterial efficacy of 0.12% chlorhexidine and 50 and 75% essential oil of Matricaria recutita on Porphyromonas gingivalis and Prevotella intermedia at 24 and 48 hours. Clinical significance: The study demonstrates that essential oil derived from Matricaria recutita may effectively combat bacteria associated with periodontal disease. This discovery has the potential to impact dental practice by introducing a natural treatment option. Further research is warranted to fully elucidate the clinical significance and potential applications of this finding.