Antimicrobial peptides (AMPs) have shown rapid and potent effect against planktonic bacteria. However, control of periodontopathic biofilms is a challenge even for AMPs. Thus, the present study evaluates in vitro antimicrobial activity of synthetic peptide LyeTxI and association compound LyeTxI/β-cyclodextrin (βCD) against multispecies biofilms. Sensibility to LyeTxI and LyeTxI/βCD was determined for planktonic Gram-negative periodontopathogens. Time-kill kinetic assay was performed at minimum inhibitory concentrations (MICs) in all planktonic strains. Multispecies biofilms were grown on pegs using a biofilm device and studied by scanning electron microscopy at 2, 5, and 10 days. Minimal biofilm eradication concentration (MBEC) was determined for 2- and 4-day multispecies biofilms. Metabolic activity of biofilms was determined by fluorometry study. Biofilms showed reproducible cell density on pegs of the biofilm device. LyeTxI and LyeTxI/βCD were active against all strains tested at concentrations ≤62.5 μg/mL. Kinetic assays showed rapid bactericidal effect of LyeTxI against all periodontopathogens. MBECs of LyeTxI and LyeTxI/βCD against multispecies 2-day biofilms were two-fold higher than MICs of cells shed from biofilms. LyeTxI was able to reduce multispecies 2-day metabolic activity by 90%. Multispecies 4-day biofilms were tolerant to all agents tested. LyeTxI and LyeTxI/βCD are active against periodontopathic bacteria, showing rapid bactericidal effect and may be used to prevent biofilm development. In the future, AMPs could be therapeutic tools for treatment of periodontitis.