Food spoilage is a serious problem in the food industry, since it leads to significant economic losses. One of its main causes is the cross-contamination of food products from industrial surfaces. Three spoilage bacterial species which are highly present in meat and the gastrointestinal tract of chickens were selected: Pseudomonas fragi, Leuconostoc gasicomitatum, and Lactobacillus reuteri. The dual aim was to determine their ability to form monospecies biofilms and to examine how they interact when they coexist together. To do so, mature monospecies biofilms were produced statically for seven days at a temperature of 30 ◦C. L. gasicomitatum was also used to investigate the behavior of P. fragi and L. reuteri in the formation of multispecies biofilms. The structure and composition of the monospecies biofilms were evaluated by direct epifluorescence microscopy, and the multispecies biofilms were evaluated by plate counting. Both L. gasicomitatum and L. reuteri were able to form biofilms, with counts of approximately 7 Log CFU/cm2 and a defined structure. However, P. fragi obtained counts to the order of 4 Log CFU/cm2 , which is significantly different from the previous species (P < 0.05), and it had no network of cell conglomerates. The content of the L. gasicomitatum and L. reuteri biofilm matrices were 70-80% protein, unlike P. fragi, which presented a higher polysaccharide content (P < 0.05). In the multispecies biofilms, the presence of P. fragi did not affect the growth of L. gasicomitatum, which remained at between 5.76 to 6.1 Log CFU/cm2 . However, L. reuteri was able to displace L. gasicomitatum growth after 24 h of coexisting in a mixed biofilm, presenting differences in counts of approximately 2 Log CFU/cm2 . The study of the biofilms constructed by food industry resident microbiota can help to understand the ecological relations that exist between species, characterize them, and propose strategies to eliminate them. The name of genes and species should be written in italic.