Understanding and predicting bacterial behaviour in foods is vital for food safety. Although in the past most food microbiological research was conducted in liquids, it is now known that bacterial behaviour changes fundamentally when grown in structured environments. Furthermore, the bacterial behaviour is also affected by the natural microflora of foods and possible cross-contaminants. These can interact synergistically but can also be used as an antagonistic tool for food safety. The aim of this work is to perform a systematic study on the impact of fat concentrations (FC) on bacterial kinetics, their inter-species interactions, and their sensitivity towards the mild preservation technology of cold atmospheric plasma (CAP) in complex triphasic 3D systems. Building on our biphasic protein/polysaccharide food model (FM), a third fat phase was incorporated into the system (10-60%). Single-cultures of Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis were grown on the surface of the FMs, as well as listerial co-cultures with each of the listed bacterium. A multiscale analysis took place macroscopically (plate count) and microscopically (confocal-laser-scanning-microscopy). Subsequently, the single- and co-cultures grown on the FMs surfaces were treated with CAP. Overall, the macroscopic analysis revealed increased growth of single cultures in comparison to co-cultures, but no significant impact in respect to the tested FCs. However, on the microscopic scale, generally, differences between the FCs were observed. More specifically, the bacterial colony sizes and biofilm formation were increased with increasing FC, more significant in co-cultures than in single-cultures. Due to these microscopic differences, a different level of cell-to-cell and colony-to-colony interaction takes place. This was further demonstrated by the susceptibility and resistance of the single- and co-cultures to the CAP treatment. In conclusion, our results indicate the importance of accounting for the microflora complexity and their interactions in food systems when predicting microbial behaviour and designing food decontamination treatments to ensure food safety.