The use of chemical antimicrobials to control undesirable microbial agents contributes to the rise of antimicrobial resistance (AMR) and environmental pollution. In alignment with the One Health paradigm, there is a growing emphasis on developing innovative alternatives, such as harnessing beneficial microorganisms with strong competitive traits. This approach aims to sculpt the microbial ecology of surfaces, ultimately restraining the proliferation of undesirable microorganisms. In this contribution, we introduce a novel methodology that enables a rational and systematic selection and combination of strains for such applications. Our approach leverages live-cell fluorescent imaging through High Content Screening Confocal Laser Scanning Microscopy (HCS-CLSM). This method was used to assess a collection of Bacillus species strains in diverse multi-species biofilm scenarios, aiming to understand their impact on the growth of different bacterial pathogens and unravel the nature of the mechanisms underlying their exclusion. Our study results reveal the exceptional antagonistic influence of three Bacillus velezensis strains against pathogens. Moreover, these strains demonstrated the ability to form a stable kin-compatible consortium, resulting in enhanced surface colonisation of the biofilm compared to individual strains. By leveraging live-cell imaging kinetics and modelling, we were able to demonstrate Jameson nutritional competition in most explored scenarios. Additionally, regarding antagonist interactions between B. velezensis against Enterococcus cecorum, our modelling revealed a Lotka-Volterra prey-predator dynamic, indicating an active interference mechanism. Furthermore, we observed a notable increase in the inhibition of Salmonella enterica serovar Enteritidis when B. velezensis was pre-established on the surface, suggesting a disctinct biofilm-associated exclusion effect. This methodology establishes an innovative approach for constructing robust positive biofilms with potent effects against pathogens.