Background and Objectives: Bacteria and its remnants beneath the restorations predispose the tooth to secondary caries and pulpal pathology. Hence, various chemical antibacterial agents are suggested to disinfect the prepared tooth structure before the definitive restorative procedure. This study aimed to investigate the effects of chemical disinfectant solutions on the micro-shear bond strength (µSBS) and microhardness of total-etch and self-etch resin-infiltrated human dentin. Materials and Methods: 100 caries-free intact permanent third molar teeth were vertically sectioned into the buccal and lingual half. All these specimens were mounted on acrylic resin and underlying dentin surfaces were exposed by grinding. Samples were randomly divided into five groups [n = 20] following total-etch and self-etch adhesive protocol. Teeth samples were divided according to surface treatment, as Group I (Control-CNT), Group II (2% chlorhexidine-CHX), Group III (5.25% sodium hypochlorite-NaOCl), Group IV (17% ethylenediaminetetraacetate acid—EDTA) and Group V (10% povidone iodine-PVI). A randomly selected 10 samples from each subgroup were used for µSBS and microhardness tests. After surface treatment and bonding procedure, nono-hybrid composite cylinders with a 3-mm diameter and 2-mm height were directly cured over the dentin substrate. The samples for µSBS were subjected to 5000 thermocycles and tested using a universal testing machine. Microhardness was assessed using a micro-indenter instrument, data were statistically analyzed using a one-way analysis of variance and Tukey HSD tests at p < 0.05. Results: Amongst the chemical disinfectant assessed, 2% CHX did not affect µSBS and produced a marginal reduction in dentin microhardness compared to the control group. The 5.25% NaOCl and 17% EDTA significantly compromised the microhardness of the dentin substrate. Meanwhile, 10% PVI surface treatment resulted in a substantial reduction in µSBS between composite and dentin. Conclusions: CHX with preservation of bonding to dentin and insignificant negative effect on dentin microhardness is a safe option for tooth disinfection.