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Background: Avocado is affected by Colletotrichum gloeosporioides causing anthracnose. Antagonistic microorganisms against C. gloeosporioides represent an alternative for biological control. Accordingly, in the present study, we focused on the isolation and characterization of potential antagonist bacteria against a member of the C. gloeosporioides species complex with respect to their possible future application., Results: Samples of avocado rhizospheric soil were aquired from an orchard located in Ocuituco, Morelos, Mexico, aiming to obtain bacterial isolates with potential antifungal activity. From the soil samples, 136 bacteria were isolated and they were then challenged against a member of the C. gloeosporioides species complex; only three bacterial isolates A1, A2 and A3 significantly diminished mycelial fungal growth by 75%, 70% and 60%, respectively. Two of these isolates were identified by 16S rRNA as Bacillus mycoides (A1 and A2) and the third was identified as Bacillus tequilensis (A3). Bacillus mycoides bacterial cell-free supernatant reduced the mycelial growth of a member of the C. gloeosporioides species complex isolated from avocado by 65%, whereas Bacillus tequilensis A3 supernatant did so by 25% after 3 days post inoculation. Bacillus tequilensis mycoides A1 was a producer of proteases, indolacetic acid and siderophores. Preventive treatment using a cell-free supernatant of B. mycoides A1 diminished the severity of anthracnose disease (41.9%) on avocado fruit., Conclusion: These results reveal the possibility of using B. mycoides A1 as a potential biological control agent. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)