| Summary: | This study aims to investigate the mono- and dual-species biofilm formation of <i>Listeria monocytogenes</i> and <i>Pseudomonas aeruginosa</i> incubated in different culture mediums, inoculum ratios, and incubation time. The planktonic cell population and motility were examined to understand the correlation with biofilm formation. The results showed that chicken juice significantly inhibited the biofilm formation of <i>L. monocytogenes</i> (<i>p</i> < 0.05). <i>Pseudomonas aeruginosa</i> was the dominant bacteria in the dual-species biofilm formation in the trypticase soy broth medium. The dynamic changes in biofilm formation were not consistent with the different culture conditions. The growth of planktonic <i>L. monocytogenes</i> and <i>P. aeruginosa</i> in the suspension was inconsistent with their growth in the biofilms. There was no significant correlation between motility and biofilm formation of <i>L. monocytogenes</i> and <i>P. aeruginosa</i>. Moreover, scanning electron microscopy (SEM) results revealed that the biofilm structure of <i>L. monocytogenes</i> was loose. At the same time, <i>P. aeruginosa</i> formed a relatively dense network in mono-species biofilms in an initial adhesion stage (24 h). SEM results also showed that <i>P. aeruginosa</i> was dominant in the dual-species biofilms. Overall, these results could provide a theoretical reference for preventing and controlling the biofilm formation of <i>L. monocytogenes</i> and <i>P. aeruginosa</i> in the food processing environment in the future.
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