Complementary solutions to chemical inputs are being implemented in agricultural environments to reduce their application. The preventive use of synthetic microbial communities (SynComs) forming beneficial biofilms on the surfaces of livestock facility buildings is being developed to limit the establishment of undesirable bacteria by competitive exclusion (https://doi.org/10.1016/j.bioflm.2022.100075). In this study, we present a dataset integrating live-cell imaging of multi-strain biofilms across diverse scenarios (https://doi.org/10.1016/bs.mim.2023.03.002). Through this method, we were able to identify beneficial strains based on their superior ability to exclude undesirable bacteria and form mixed biofilms. Multi-strains biofilms were observed using a Leica SP8 AOBS inverted laser scanning microscope (CLSM, LEICA Microsystems, Wetzlar, Germany) at the INRAE MIMA2 platform (https://doi.org/10.15454/1.5572348210007727E12). All the experiments consisted in interacting a bacterium genetically labelled with GFP with another bacterium in spatially organized biofilms. The entire population was labelled with SYTO61 (Invitrogen, Carlsbad, CA, USA; a cell-permeant red dye that labels nucleic acid) dual labelling. In this system, cells labelled only in red correspond to the non-genetically labelled strains, and cells labelled both in red and green correspond to the genetically tagged ones. GFP excitation was performed at 488 nm with an argon laser, and the emitted fluorescence was recorded within the ranges 500-550 nm. SYTO 61 was excited with a helium-neon laser at 633 nm and the emitted fluorescence was collected with a hybrid detector in the range of 650-750 nm. The acquired images had a resolution of 512 × 512 pixels, corresponding to a physical area of 184.52 µm x 184.52 µm and a pixel size of 0.361 µm. The first dataset corresponding to 3168 stacks of images represents the interactions between Bacillus spp. or Pediococcus spp. with GFP-labelled pathogenic bacteria (Staphylococcus aureus, Enterococcus cecorum, Escherichia coli, Salmonella enterica serovar Enteritidis). The second dataset corresponding to 756 stacks of images represents interactions between GFP-labelled B. velezensis and other Bacillus spp. or Pediococcus spp. For every experiment, three biological replicates were carried out with 4 technical replicates each, representing 12 technical values per condition.