Four mushroom (Flammulina velutipes) production plants were sampled to investigate the prevalence and contamination source of Listeria monocytogenes. Among 295 samples, the prevalence of L. monocytogenes was 18.6%; the contamination appeared to originate from the mycelium-scraping machinery, contaminating both the product and upstream packaging equipment. Of 55 L. monocytogenes isolates, lineages I.1 (1/2a-3a) and II.2 (1/2b-3b-7) accounted for 65.5% and 34.5%, respectively. In addition, lineage I.1 formed significantly thicker biofilms than those within lineage II.2, as determined by crystal violet staining and scanning electron microscopy. Genotype analyses of L. monocytogenes isolates using enterobacteria repetitive intergenic consensus-polymerase chain reaction, and random amplified polymorphic DNA revealed that the surfaces of mycelium-scraping machinery may serve as the main source of L. monocytogenes contamination in three of the four plants. This study was the first report to explore the potential contamination sources of L. monocytogenes in the mushroom production chain, thereby providing baseline information for adopting prophylactic measures for critical control points during production in mushroom plants to avoid L. monocytogenes contamination.