Six environmental isolates of Staphylococcus aureus and one collection strain were investigated for their ability to form monospecies biofilms and dual species biofilms with Listeria monocytogenes EGD-e on stainless steel coupons. All isolates were able to grow as biofilms but their ability to form monospecies biofilms differed. The population of L. monocytogenes EGD-e in dual species biofilms was not affected by the presence of S. aureus isolates except for strain CIP 53.156. The effect of L. monocytogenes EGD-e on the population of S. aureus was strain dependent: S. aureus population either increased or decreased or was not affected in the presence of L. monocytogenes EGD-e in dual species biofilms. Dual species biofilms were grown with L. monocytogenes EGD-e and the strain CIP 53.156 of S. aureus on stainless steel coupons under batch and dynamic conditions. Higher sessile populations of L. monocytogenes EGD-e were observed in the presence of S. aureus CIP 53.156. Microscope observations by scanning electron microscopy (SEM) revealed an intimate association of L. monocytogenes EGD-e and S. aureus CIP 53.156 in dual species biofilms. An increase of the number of L. monocytogenes EGD-e cells was observed in the presence of S. aureus CIP 53.156 cell-free supernatant. This activity was retained after ultrafiltation (<3 kDa), was heat stable but was lost after proteinase K treatment.