Background and aim: In dental treatments, use of antibiotic carriers can decrease microorganisms more efficiently. In this study, gentamicin (GEN) was loaded onto poly lactic-co-glycolic acid (PLGA) copolymers and PLGA-nano hydroxyapatite (nHA) composite microspheres for a controlled release. The amount of drug release was measured for 20 days. Materials and methods: In this in-vitro study, microspheres were prepared using the water/oil/ water technique and different concentration of GEN loaded onto PLGA copolymers and PLGA- nHA composite microspheres. Loaded microspheres were evaluated morphologically using scanning electron microscopy (SEM). The rate of drug release from the composite microspheres was measured in phosphate buffered saline (PBS) medium for a period of 20 days using UV spectrophotometry (330 nm). Data were analyzed using ANOVA. Also, a microbial culture was carried out of microspheres with the least amount of drug in the first and last day of drug release assessment. Results: SEM images showed that the microspheres had a smooth surface and the pattern of drug release from the PLGA copolymers and PLGA-nHA composite microspheres loaded with GEN was different in each group at different time points, but this difference only in the PLGA+0.02 GEN group was significant. There was a significant difference between this group and other groups in the amount of released drug at day 6. Also, the results of microbial culture of the group with 0.02 GEN showed the antibacterial effect of these microspheres in the last day of experiment. Conclusion: The release profiles observed in this study and the well-established biocompatibility of PLGA indicate that the composite microspheres used in this study are suitable for infection control purposes in dentistry.