Background Chronic periodontitis is a multifactorial infectious disease, where multiple bacteria, such as Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum are implicated. The main purpose of researching natural products is to find substances or compounds with antimicrobial activity. Aim The objective of this work was to determine antimicrobial activity from extracts and obtained fractions from Piper marginatum Jacq and Ilex guayusa Loes on P. gingivalis ATCC 33277, F. nucleatum ATCC 25586, and P. intermedia ATCC 25611. Methods Total ethanol extracts were obtained from both plants. Fractions were obtained from total ethanol extracts with amberlite as a stationary phase employing hexane, acetone, and ethanol-water as solvents. Qualitative and quantitative phytochemical characterization was performed on total ethanol extracts from both plants. Antimicrobial activity from total ethanol extracts and fractions from both plants were evaluated on P. gingivalis ATCC 33277, F. nucleatum ATCC 25586, and P. intermedia ATCC by the well diffusion method with Wilkins–Chalgren agar. Results Piper marginatum Jacq total ethanol extract presented antimicrobial activity against all three bacteria, whereas Ilex guayusa Loes was only efficient against P. gingivalis ATCC 33277 and P. intermedia ATCC 25611, with inhibition halos from 9.3 to 30 mm. Ilex guayusa Loes obtained fractions presented antimicrobial activity against all three microorganisms evaluated, with inhibition halos ranging from 9.7 to 18.7 mm. In regards to Piper marginatum Jacq fractions, inhibition halos were between 8.3 and 19 mm, against all three microorganisms evaluated; only hexane fraction did not present antimicrobial activity against F. nucleatum ATCC 25586. Conclusion Piper marginatum Jacq and Ilex guayusa Loes total ethanol extracts and fractions presented outstanding antimicrobial activity against P. gingivalis ATCC 33277, P. intermedia ATCC 25611, and F. nucleatum ATCC 25586.