Aims: The isolation and characterization of a novel coffee-associated Bacillus mojavensis strain, designated as strain AB1, and its survival on the coffee phyllosphere. Methods and Results: A pair of 16S rDNA primers was designed to amplify a highly variable region within the 16S rDNA gene of Bacillus spp., with the purpose of identifying the AB1 isolate through PCR and sequence analysis. By this method, AB1 was identified as a strain of B. mojavensis. Bioassays were carried out to characterize the broad spectrum antifungal activity of AB1. Plant colonization studies revealed that AB1 could colonize the coffee phyllosphere better than Bacillus thuringiensis. Conclusions: These studies suggest that AB1 could be a new strain of B. mojavensis. AB1 is also shown to have antifungal activity against a wide spectrum of pathogenic fungi. The antifungal metabolite of AB1 has been partially characterized as a thermostable, protease- and alkali-resistant substance that is secreted into the surrounding medium. Significance and Impact of the Study: As far as is known, this is the first strain of B. mojavensis which has been identified as inhabiting the coffee phyllosphere. The study highlights the potential use of AB1 as an antifungal agent in the coffee crop and as a delivery agent of the insecticidal toxin of B. thuringiensis to the coffee phyllosphere. The 16S rRNA identification strategy discussed could also be used in the identification of other new Bacillus strains.