The biodegradable chitosan microspheres containing vancomycin hydrochloride (VANCO) were prepared by spray drying method with different polymer:drug ratios (1:1, 2:1, 3:1 and 4:1). Thermal behaviour, particle size and distribution, morphological characteristics, drug content, encapsulation efficiency, in vitro release assessments of formulations have been carried out to obtain suitable formulation which shows sustained-release effect when implanted. Sterilized VANCO loaded microspheres were implanted to proximal tibia of rats with methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. Intramuscular (IM) injection of VANCO for 21 days was applied to another group for comparison. After 3 weeks of treatment, bone samples were analysed with a microbiological assay. According to the results, encapsulation efficiency and yield of microspheres in all formulations were higher than 98% and 47%, respectively. Particle sizes of microspheres were smaller than 6 microm. All microsphere formulations have shown sustained-release effect. In vitro drug release rate decreased due to the increase in polymer:drug ratio but no significant difference was seen between these results (p>0.05). Based on our in vivo data, rats implanted VANCO-loaded chitosan microspheres and administered IM injection showed 3354+/-3366 and 52500+/-25635 colony forming unit of MRSA in 1g bone samples (CFU/g), respectively. As a result, implanted VANCO-loaded microspheres were found to be more effective than IM route for the treatment of experimental osteomyelitis.