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The genus Bacillus has been isolated from a wide range of environments and comprises species with biotechnological applications, such as bioinseticides, biofungicides, and biofertilizers. This study investigated the endophytic occurrence and diversity of Bacilli in the apoplastic fluid of sugarcane cultivated in Brazil. Their biocontrol activity against fungi and their plant growth promotion (PGP) potential in sugarcane mini-set plants were also evaluated. Forty-two Bacillus colonies were isolated from the apoplastic fluid of one-year-old commercial sugarcane varieties and analyzed by sequencing the 16S rRNA gene. The ability to produce indols and siderophores and to solubilize phosphate and zinc were evaluated in vitro , along with their cellulose and protease activity and, antagonistic activity against sugarcane phytopatogenic fungi. The PGP potential was determined using strains which were inoculated in sugarcane mini-set plants grown in sterile substrate and later in unsterilized soil pots. The 16S rRNA analyses demonstrated high bacterial diversity with the majority closely related to B. megaterium followed by B. aryabhattai and B. cereus. B. pumilus , B. velezensis , B. safensis , B. flexus and Paenibacillus taichungensis species were also identified. Most strains were able to produce indol, solubilize zinc oxide and presented cellulase activity, while phosphate solubilization and siderophore production was observed in only few strains. The strains LMC21, LMC44 and LMC48 presented high biocontrol activity against the sugarcane fungi Colletotrichum falcatum. The spore formation was observed for strains grown for 8 days in the HCT medium but not in presence of sugarcane juice. Some strains also accumulated higher biomass in sugarcane (sterile conditions) and a mixture of three Bacillus strains performed better in the soil-pot inoculation experiment. These findings show for the first time that many Bacillus species are present in the apoplastic fluid of sugarcane grown in Brazil and indicate that some strains have PGP and biocontrol potential for application as biofertilizer in sugarcane plants. • Apoplast fluid of sugarcane plants as ecological niche for Bacillus species. • Bacillus species presented high biological activity against sugarcane fungi Colletotrichum falcatum. • A Bacillu s inoculant consortium stimulates growth of sugarcane plants. [ABSTRACT FROM AUTHOR]