The structural integrity of a dentin matrix that has been demineralized by the clinical use of etchants or calcium-depleting endodontic irrigants, such as endodontic ethylenediaminetetraacetic acid (EDTA), is often deteriorated due to the collagenolytic activities of reactivated endogenous enzymes as well as the infiltration of extrinsic bacteria. Therefore, the biomodification of dentin collagen with improved stability and antibacterial activity holds great promise in conservative dentistry. The purpose of this study was to evaluate the effects of the combined application of trimethylated chitosan (TMC) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) on the biostability and antibacterial activity of the demineralized dentin collagen matrix. The morphological changes in the collagen matrix were observed by scanning electron microscopy (SEM), the amount of TMC adsorbed on the collagen surface was detected by X-ray photoelectron spectroscopy, and the elastic modulus was measured by a three-point bending device. Dry weight loss and amino acid release were detected to evaluate its anti-collagenase degradation performance. The antibacterial performance was detected by confocal microscopy. The TMC-treated group had less collagen space and a more compact collagen arrangement, while the untreated group had a looser collagen arrangement. The combined application of TMC and EDC can increase the elastic modulus, reduce the loss of elastic modulus, and result in good antibacterial performance. The current study proved that a dentin collagen matrix biomodified by TMC and EDC showed improved biodegradation resistance and antibacterial activities.