[Objective] Many environmental factors, such as carbon sources, regulate the biosynthesis of antimicrobial compounds and influence the biocontrol capacity of Pseudomonas fluorescens. P. fluorescens 2P24 protects various crop plants against root diseases caused by plant pathogens. Among a range of antimicrobial compounds secreted by 2P24, 2,4-diacetylphloroglucinol (2,4-DAPG) is the major determinant of its biocontrol potential. This study investigated the impact of exposing strain 2P24 to selected carbon sources on the production of 2,4-DAPG. [Methods] Antifungal activity of strain 2P24 was tested on potato infusion agar with different carbon sources against Rhizoctonia solani. The reporter strain 2P24 (p970Gm-phlAp) was subjected to a random mini-Tn5 insertion mutagenesis. The collection of Tn5 insertion mutants was then screened for improved phlA expression on potato infusion agar with fructose. [Results] Strain 2P24 cultured on potato infusion with glucose strongly inhibited the growth of R. solani, whereas no inhibition was observed on potato infusion agar with or without fructose. Five mutants with significantly increased phlA expression were identified and the interrupted locus in one of them was identified as the cheB gene. Genetic analysis showed that the expression of phlA and the production of phloroglucinol (PG) were strongly increased in the cheB mutant as compared with the parental strain.[Conclusion] Carbon resources influenced the expression of phlA and 2,4-DAPG production and some genetic factors involved in carbon sources to regulate the production of 2,4-DAPG.