The objective of this study was to evaluate the inhibitory effect of four natural antimicrobial compounds on the biofilm formation of Listeria monocytogenes (L. monocytogenes) isolated from beef processing plants in China. Firstly, biofilm-forming ability of the isolated L. monocytogenes (n = 131) was investigated. Furthermore, the biofilm inhibitory assay of four natural antimicrobial compounds (cinnamaldehyde, eugenol, resveratrol and thymoquinone) was tested at subminimum inhibitory concentrations (sub-MICs), with sodium hypochlorite as a control. The results showed that all 131 tested strains produced biofilms, and 67.2% (n = 88) were strong biofilm producers. The strongest biofilm formation capacity occurred at 25 °C and pH 7.4, and the biofilm-forming ability of L. monocytogenes is specific at the genotype level. Four natural antimicrobial compounds were effective at inhibiting biofilm formation of L. monocytogenes at sub-MIC (1/4 × MIC and 1/8 × MIC), and the effective inhibitory concentrations of resveratrol and thymoquinone were as low as 25 μg/mL and 6.25 μg/mL. Scanning electron microscopy (SEM) showed distinguishable inhibitory effect of natural antimicrobial compounds on biofilm formation. These findings indicate a potential application of natural antimicrobial compounds in the prevention of L. monocytogenes biofilm.