The objective of this study was to develop a chitosan-based biomaterial with calcium hydroxide and 2% chlorhexidine for intracanal treatment application and, consequently, to diminish the number of microorganisms in the root canal system. The chitosan solution was prepared by dissolving it in 2% and 4% acetic acid (v/v) for 1 h at room temperature (25 °C) with magnetic agitation (430 rpm). Calcium hydroxide was obtained in two stages: the first was the synthesis of the calcium oxide—CaO, and the second was that of the calcium hydroxide—Ca(OH)2. The samples were developed using different concentrations of chitosan, calcium hydroxide, and chlorhexidine 2%. They were codified as Ca(OH)2 + Q2% (M1), Ca(OH)2 + Q4% (M2), Ca(OH)2 + Q2% + CLX (M3), Ca(OH)2 + Q4% + CLX (M4), Ca(OH)2 + Q2% + PEG (M5), and Ca(OH)2 + Q4% + PEG (M6). They were characterized through Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and rheological measurement, and the antimicrobial activity was evaluated in vitro. Characteristic absorption bands of the source materials used in this research were observed in the FTIR spectra. The X-ray diffraction technique indicated that the material has a semi-crystalline structure and that the presence of calcium hydroxide made the biomaterial more crystalline. The viscosity measurement showed a pseudoplastic behavior of the studied samples. The microbiologic analysis was positive for all samples tested, with bigger inhibition zones for the samples M3 and M4. As a result, we conclude that the formulation developed based on chitosan is promising and has potential to be an intracanal medication.