Chitosan, a natural polysaccharide sourced from crustaceans and insects, is often used with hydrogels in wound care. Evaluating its cytotoxicity and antimicrobial properties is crucial for its potential use in dentistry. Objective: To investigate the mechanical properties of gelatin hydrogels based on decaethylated chitosan and antimicrobial activity against Streptococcus mutans and their biological effects with stem cells from apical papilla (SCAPs). Material and methods: Gelatin–chitosan hydrogels were synthesized at concentrations of 0%, 0.2% and 0.5%. Enzymatic and hydrolytic degradation, along with swelling capacity, was assessed. Fourier transform infrared spectroscopy (FTIR) analysis was employed to characterize the hydrogels. The interaction between hydrogels and SCAPs was examined through initial adhesion and cell proliferation at 24 and 48 h, using the Thiazolyl Blue Tetrazolium Bromide (MTT assay). The antimicrobial effect was evaluated using agar diffusion and a microdilution test against S. mutans. Uniaxial tensile strength (UTS) was also measured to assess the mechanical properties of the hydrogels. Results: The hydrogels underwent hydrolytic and enzymatic degradation at 30, 220, 300 min and 15, 25, 30 min, respectively. Significantly, (p < 0.01) swelling capacity occurred at 20, 40, 30 min, respectively. Gelatin–chitosan hydrogels’ functional groups were confirmed using vibrational pattern analysis. SCAPs proliferation corresponded to 24 h = 73 ± 2%, 82 ± 2%, 61 ± 6% and 48 h = 83 ± 11%, 86 ± 2%, 44 ± 2%, respectively. The bacterial survival of hydrogel interaction was found to be 96 ± 1%, 17 ± 1.5% (p < 0.01) and 1 ± 0.5% (p < 0.01), respectively. UTS showed enhanced (p < 0.05) mechanical properties with chitosan presence. Conclusion: Gelatin–chitosan hydrogels displayed favorable degradation, swelling capacity, mild dose-dependent cytotoxicity, significant proliferation with stem cells from apical papilla (SCAPs), substantial antimicrobial effects against S. mutans and enhanced mechanical properties. These findings highlight their potential applications as postoperative care dressings.