**1. Introduction**

Surfaces in the food industry provide an excellent substrate for the development of biofilms by spoilage and pathogenic bacteria [1]. These biofilms are typically highly persistent and can increase the likelihood of cross-contamination, potentially leading to food deterioration and/or serious food-borne diseases [2–5].

Biofilms are heterogeneous in vivo, comprising different microorganisms that interact with each other and form a complex multi-species community. Cooperation or competition occurs between the species and it shapes the community by influencing attachment, microcolony formation, and/or resistance to stress conditions [6–10]. Multi-species biofilms may exhibit enhanced fitness and react differently to antimicrobials from monocultures and planktonic cells [11].

Contamination with *Listeria monocytogenes* causes a large number of food poisoning outbreaks annually [12]. According to a study performed by the European Food Safety Authority in 2018, this food-borne pathogen is the leading cause of hospitalization and death in Europe [13]. Listeriosis is frequently associated with fish and fishery products, ready-to-eat salads, and different meat products. Paté, butter, and soft and semi-soft cheeses are also potential carriers of the pathogen [13,14]. Its persistence under different environmental stresses (e.g., low temperature) makes it difficult to eradicate and cross-contamination risk is high [15]. *Staphylococcus aureus*, *Escherichia coli,* and *Pseudomonas putida* strains are also associated with food spoilage and food poisoning outbreaks [16,17]. Moreover, certain food-spoilage bacteria such as *P. putida* can enhance the adhesion, colonization, and biofilm formation of *L. monocytogenes*, whereas others such as *Staphylococcus sciuri* can inhibit it [18,19]. Although the chemical preservatives that are permitted in foods are considered to not cause any side effects, concerns have been raised about the safety of nitrites and sulphites. To date, no conclusive evidence that nitrite is directly carcinogenic has been provided, but at high doses it has been suggested to be a co-carcinogen [20,21].

Against this background, there is increasing demand for healthier products and natural food additives. To meet these demands, researchers have started to examine natural preservatives instead of synthetic ones [22]. Plants are a source of a range of substances with antimicrobial properties, which are promising candidates for the development of new anti-infective agents [23]. Among these, essential oils (EOs) from aromatic and medicinal plants have been a focus of attention in recent decades [24–27]. In addition, the capacity of some EOs to inhibit biofilm formation in mono- and polymicrobial systems has been documented, suggesting their potential utilization as food preservatives and sanitizing agents [28–30]. In this context, targeting polymicrobial cultures with EOs might be effective for reducing the growth and activity of food-related pathogens.

In previous studies, lemon, marjoram, and cinnamon EOs showed inhibitory effects against *E. coli* and *P. putida* biofilms in mixed-culture systems [31,32]. Here the influence of cinnamon, marjoram, and thyme EOs and their major components, namely, trans-cinnamaldehyde, terpinen-4-ol, and thymol, respectively, on the formation of *L. monocytogenes*, *E. coli*, *S. aureus,* and *P. putida* mono- and polymicrobial biofilms was investigated.

#### **2. Materials and Methods**
