Cholesterol has been widely used in drug delivery systems including implant. Doxycycline hyclate (DH)-loaded cholesterol in situ forming gels using N-methyl pyrrolidone as a solvent were prepared and investigated for their properties including viscosity, rheology, syringeability, gel formation, drug release, degradation and antimicrobial activities. The burst drug release of a DH-loaded in situ forming gel using cholesterol as the gelling agent was minimized when the amount of benzyl benzoate was increased. The viscosity of the system was increased as the amount of benzyl benzoate was increased with Newtonian flow. The systems were easy to inject into the target site because of their minimal force of syringeability. They could transform from solution into matrix-like structures, but formulations with higher concentrations of benzyl benzoate took a longer time. However, the degradability was decreased when the amount of benzyl benzoate was increased. These systems inhibited P. gingivalis, S. mutans and S. aureus effectively. DH-loaded cholesterol in situ forming gel system comprising 10% benzyl benzoate was the most suitable owing to its sustainable release manner for 10 days and therefore was the proper formulation for periodontitis treatment.