The increase in human consumption of animal proteins combined with public awareness towards sustainability and animal welfare implies changes in the management of meat production. This is followed by increasingly restrictive regulations on antimicrobial products such as chemical biocides and antibiotics, used in particular to control pathogens that can spread zoonotic diseases. Aligned with the One Health concept, alternative microbial solutions are under development and are starting to be used in animal production facilities. Beneficial bacteria able to form positive biofilms and guide surface microbial ecology to limit microbial pathogen settlement in animal housing and equipment surfaces are promising tools that could complement existing biosecurity practices to maintain the hygiene of livestock buildings. Although the benefits of these positive biofilms have already been documented at farm level, the associated fundamental mechanisms and the rationale of the microbial composition of these new products are still sparse. Here, we provide an overview of the envisioned modes of action of positive biofilms used on livestock building surfaces. We present a pipeline for rational strains selection for such applications based on genome and phenotype analysis using High Content Screening-Confocal Laser Scanning Microscopy (HCS-CLSM) combined with genetically engineered fluorescent strains and image analysis. This pipeline was applied to screen a collection of Bacillus spp. for their effect on the growth of several bacterial pathogens in different mixed biofilms models and to decipher the mechanisms of bacterial pathogens exclusion. The ability of Bacillus spp. to form stable multi-species biofilms will be studied in order to assemble positive consortia with enhanced ability to colonize surfaces and exclude pathogens.