Pectolytic strains of Pseudomonas fluorescens are opportunistic pathogens of broccoli, causing head rot in temperate regions of the world. In this study, we investigated the potential of two bacterial isolates, P. fluorescens m6418 and Bacillus sp. A24, for biological control of broccoli head rot caused by P. fluorescens 5064, isolated from diseased broccoli in Scotland, UK. P. fluorescens m6418, a Tn5 mutant of wild-type 5064, is nonpathogenic and overproduces an extracellular metabolite with strong antimicrobial activity. In this study, we identified the anti-microbial metabolite produced by strain m6418 as pyrrolnitrin. P. fluorescens m6418 had significant inhibitory effects against strain 5064 both in culture and on broccoli leaves. In an excised broccoli head pathogenicity test, strain m6418, when coinoculated with P. fluorescens 5064, reduced disease by 41%. Bacillus sp. A24 produces an enzyme that can degrade N-acyl homoserine lactones, signaling molecules employed by bacteria for quorum sensing. Bacillus sp. A24 was capable of out-competing P. fluorescens 5064 when grown together in culture, and could degrade the quorum sensing signal of P. fluorescens 5064 (and thereby attenuate its virulence gene production). However, Bacillus sp. A24 had only a limited biocontrol effect on P. fluorescens 5064 in the excised broccoli head assay.