AbstractBiocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp., responsible for potato soft rot disease. Discovering agents that can effectively control both fungal and bacterial pathogens in potatoes has always presented a challenge. Biological controls were investigated using 500 bacterial strains isolated from rhizospheric microbial communities, along with two promising biocontrol strains: Pseudomonas (T17-4 and VUPf5). Bacillus velezensis (Q12 and US1) and Pseudomonas chlororaphis VUPf5 exhibited the highest inhibition of fungal growth and pathogenicity in both laboratory (48%, 48%, 38%) and greenhouse (100%, 85%, 90%) settings. Q12 demonstrated better control against bacterial pathogens in vivo (approximately 50%). Whole-genome sequencing of Q12 and US1 revealed a genome size of approximately 4.1 Mb. Q12 had 4413 gene IDs and 4300 coding sequences, while US1 had 4369 gene IDs and 4255 coding sequences. Q12 exhibited a higher number of genes classified under functional subcategories related to stress response, cell wall, capsule, levansucrase synthesis, and polysaccharide metabolism. Both Q12 and US1 contained eleven secondary metabolite gene clusters as identified by the antiSMASH and RAST servers. Notably, Q12 possessed the antibacterial locillomycin and iturin A gene clusters, which were absent in US1. This genetic information suggests that Q12 may have a more pronounced control over bacterial pathogens compared to US1. Metabolic profiling of the superior strains, as determined by LC/MS/MS, validated our genetic findings. The investigated strains produced compounds such as iturin A, bacillomycin D, surfactin, fengycin, phenazine derivatives, etc. These compounds reduced spore production and caused deformation of the hyphae in F. solani. In contrast, B. velezensis UR1, which lacked the production of surfactin, fengycin, and iturin, did not affect these structures and failed to inhibit the growth of any pathogens. Our findings suggest that locillomycin and iturin A may contribute to the enhanced control of bacterial pectolytic rot by Q12.