Bacillus amyloliquefaciens K103 isolated from a lemon sample was used as a biocontrol agent to suppress Rhizoctonia solani Kühn and other fungal plant pathogens. Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis indicated that the antifungal compounds were two isomers similar to bacillomycin L. One of the predominant active fractions was subjected to quadrupole time-of-flight mass spectrometry and amino acid analysis to determine its structural characteristics, revealing that the antifungal compound with a molecular mass of 1,034.5464 was identical to bacillomycin L. This is the second report of lemon microflora producing bacillomycin L or any antifungal compound, suppressing the growth of R. solani Kühn. Meanwhile, the study provided insights into the enormous potential of food microbial resources and bacillomycin L antibiotics in biological control and sustainable agriculture.