The treatment of osteomyelitis induced by Gram-negative bacilli is rarely reported in the literature. This study established a rabbit tibia model of osteomyelitis induced by the Gram-negative bacillus Escherichia coli. Using this model, pellets composed of a chitosan-bonded mixture of borate bioactive glass and gentamicin were evaluated in vitro and in vivo for the treatment of osteomyelitis induced by Escherichia coli. Our results showed that the pellets in phosphate-buffered saline released gentamicin continuously over 26 days. Without the simultaneous use of a systemic antibiotic, the implantation of the gentamicin-loaded pellets into the osteomyelitis region of the tibia resulted in the eradication of 81.82% of infections, as determined by microbiological, histological and radiographic evaluation, and supported the ingrowth of new bone into the tibia defects after 6 weeks of implantation. The results indicate that the gentamicin-loaded borate bioactive glass implant, combining sustained drug release with the ability to support new bone formation, could provide a method for treating osteomyelitis induced by Gram-negative bacilli.