Soft rot is a destructive disease that inflicts significant losses on agricultural production and the economy post-harvest. Biocontrol strategies based on antagonistic microorganisms have a broad application prospect to fight against plant pathogens. This study utilized fluorescence-activated droplet sorting (FADS) technology as an alternative to traditional plate culture methods to isolate microorganisms with antagonistic activity against the soft rot pathogen Erwinia carotovora Ecc15. Initially, the culture performance of the FADS platform was evaluated by analyzing bacterial diversity in droplet culture samples and agar plate culture samples, our data showed that droplet culture exhibited higher species richness and diversity than plate culture, and more than 95% of the operational taxonomic units (OTUs) in the droplet samples belonged to the rare biosphere. Additionally, we developed a green fluorescent protein (GFP)-Ecc15-based FADS screening system, which achieved an enrichment ratio of up to 148. Using this system, we successfully screened 32 antagonistic bacteria from rhizosphere soil sample of healthy konjac plants, and some may be novel microbial resources, including the genera Lelliottia, Buttiauxella and Leclercia. Notably, strain D-62 exhibited the strongest antibacterial ability against Ecc15, with an inhibition zone diameter of (20.86 ± 1.56) mm. In vivo experiments conducted on the corms of Amorphophallus konjac demonstrated that strain D-62 could effectively reduce the infection ability of Ecc15 to the corms, indicating that strain D-62 has the potential to be developed as a biocontrol agent. Our findings suggested that the FADS screening system showed a screening efficiency approximately 3 × 103 times higher than plate screening system, while significantly reducing costs of infrastructure, labor and consumables, it provides theoretical guidance for the screening of other plant pathogen biocontrol bacteria.