Fusobacterium nucleatum (Fn), a common conditioned oral pathogen, has been found to be able to protect colon, breast and lung cancer cells against antibiotics and promote their proliferation and metastasis. The purpose of this study was to explore the inhibitory effect and mechanism of L-3-phenyllactic acid on Fn to provide directions for cancer prevention through the consumption of natural biological acids. Based on its growth curve, the optimum growth period of Fn and the minimum inhibitory concentration (MIC) of phenyllactic acid against Fn were determined, and the changes in the morphology, integrity and damage degree of the Fn cell membrane under the action of phenyllactic acid were investigated for the purpose of evaluating the ability of phenyllactic acid to inhibit Fn. The results showed that the optimum growth period of Fn was 27 h, and that the MIC of phenyllactic acid on Fn was 0.50 mg/mL. Treatment with phenyllactic acid at the MIC for 2 h increased the cell membrane permeability and changed significantly the cell morphology of Fn. Phenyllactic acid could disrupt the biofilm formed by Fn and reduce Fn adhesion. This study confirmed that phenyllactic acid had the ability to destroy the cell membrane structure of Fn and inhibit its biofilm-forming ability, thereby leading to the leakage of cellular contents and a reduction in the intracellular protein content.