Abstract: The need to improve animal welfare, sustainability, and microbiological safety of livestock production systems is leading to changes in farm management. In line with the "One Health" concept, preventive and complementary solutions to the use of inputs such as chemical biocides and antibiotics are being developed. The application of bacteria capable of forming positive biofilms and guiding surface microbial ecology to limit the establishment of undesirable microorganisms is an emerging process to help maintain the hygiene of livestock buildings. Although the benefits of positive biofilms in the context of animal farming are increasingly established, their mode(s) of action as well as techniques to efficiently select appropriate species and/or strains to create these innovative solutions are still poorly documented. In order to contribute to the development of a new generation of products, we present an experimental approach adapted for the rational selection of species and strains for such applications. This process relies on the phenotypic analysis of the communities constituting the biofilms through confocal laser scanning microscopy (HCS-CLSM) and image analysis. This approach was used to screen a collection of candidate strains for their antagonistic properties against undesirable microorganisms, as well as to characterize interaction mechanisms within different models of mixed biofilms. The ability of candidate strains to stably coexist within mixed biofilms was studied with the aim of creating beneficial consortia with an increased capacity to colonize surfaces and to prevent/reduce the risk of colonization by undesirable microorganisms. In parallel, a sampling method based on the collection of representative material coupons from livestock building surfaces was established to characterize environmental biofilms and to study the in situ effects of applying positive biofilms. Based on these results, we initiate a discussion regarding opportunities for improvement in formulating next-generation positive biofilms. Furthermore, we examine the advantages and benefits inherent to these products as complementary elements to biosafety methods traditionally employed in livestock systems.