Aims Plant tissues are the reservoirs of beneficial and harmful microbes that regulates plant growth. In the present study, we investigated the diversity, function and colonization of sugarcane roots associated with Bacillus spp. Methods and Results A total of 20 Bacillus strains were isolated and identified by 16S rRNA gene sequencing, and their genetic diversity was examined by BOX, ERIC, REP, (GTG)(5) PCR techniques. Among all Bacillus isolates, 65% showed indole acetic acid-like compounds production, 50% solubilized phosphorus and 25% of the isolates were able to secrete siderophore. Moreover, all 20 Bacillus isolates showed antifungal activity against eight fungal pathogens and 11 of them (55%) antagonized tomato grey mold. Based on the plant growth-promoting traits and antifungal potential, isolate Y8 was selected for root and plant tissue colonization assays and a greenhouse-level sugarcane growth promotion study. Fluorescence microscopy results confirmed that isolate Y8 has a strong ability to colonize in the sugarcane root and leaves, and the root surface association of Y8 was confirmed by scanning electron microscopy. Furthermore, greenhouse experimental results demonstrated that Y8 has a significant effect on enhancing sugarcane biomass and root length. Conclusions Endophytic Bacillus strains have growth-promoting properties and anti-fungal ability that can enhance plant fitness in an eco-friendly manner. Significance and Impact of the Study Endophytic Bacillus strains would be a potential alternative to chemical fertilizer as well as a biocontrol agent in the future.