The Macrophomina phaseolina (Tassi) Goid. fungus, causal agent of charcoal root rot, is considered one of the most important pathogens in forest nurseries. Recent studies in Chile have verified the antagonistic capacity of diverse bacterial isolates against M. phaseolina in vitro as well as in field testing. The objectives of this study were to identify Bacillus isolates and establish the mechanisms of antagonism against M. phaseolina. Isolates were identified with the bioMérieux biochemical kit. Tests were done in vitro and consisted in determining the production of volatile metabolites, siderophores, and the effect of different culture temperatures (20, 25, 30, and 35 °C) on the production of diffusible metabolites and inhibition of mycelial growth of the pathogen. The isolates were identified as Bacillus subtilis IX 007, B. amyloliquefaciens VII 015 and VIII 016, B. pumilus IX 030, and B. stearothermophilus TM 008. It was not possible to identify the (-) Gram VI 009 isolate. At all the evaluated temperatures, isolates inhibited M. phaseolina, reaching 75% reduction of mycelial growth (B. subtilis IX 007 at 25 °C). Some isolates showed volatile metabolites and siderophore production as possible mechanisms of antagonism.