Aim: The objective of this in vitro study was to assess the efficacy of novel propolis-based varnish against the two conventional varnishes on quantitative and qualitative assessments of occlusion of dentin tubules and resistance to erosive and abrasive wears employing scanning electron microscope (SEM). Methods: Thirty human premolars free from caries extracted due to orthodontic reasons were included in the study. Experimental group was done based on treatment received and divided into three groups. Group A: ClinProXT Varnish (n = 10), Group B: MI Varnish (n = 10), and Group C: Propolis Varnish (n = 10) were applied. Teeth were cleaned and decoronation of crown was done with dentin disks. Dentin specimens of dimension 4 × 4 × 2 mm were prepared and subjected to finishing and polishing. The sample specimens were submersed in EDTA solution for a period of five minutes to open up the dentinal tubules. This was followed by treatment with varnishes and subjection to acidic-abrasive challenge. The specimens were analyzed with an image analyzer connected to SEM for the verification of the number of opened dentin tubules. The parameter assessed in SEM includes size, topography and surface characteristics of dentinal tubule were assessed. The obliteration potential of dentinal tubules was assessed with SEM images. Additionally, the dentin surface loss and resistance to acidic and abrasive wear were also evaluated with SEM. Data were analyzed with two-way analysis of variance (ANOVA) with post hoc Tukey's test. Results: MI Varnish caused higher obliteration of dentin tubules followed by ClinproXT Varnish. Propolis Varnish showed the least obliteration of dentinal tubules among tested experimental groups. After acidic-abrasive challenge, Propolis Varnish was found to be more efficient with less material loss among the experimental groups tested. There was an insignificant difference among the MI Varnish and ClinProXT Varnish groups. Conclusion: Simulation of hypersensitive lesions mimicking the clinical scenario was a challenging task in this in vitro study. All varnishes tested in the study had good efficacy in the management of dentin hypersensitivity (DH). Propolis-based varnish had good resistance to material loss after subjection to acidic-abrasive challenge among the tested materials. The casein phosphopeptide (CPP)-amorphous calcium phosphate (ACP)-based MI Varnish had good efficacy to obliterate the dentinal tubules among the tested materials. It was prudent to select the varnishes with good long-term efficacy to survive in the clinical scenario which still remains a challenging task for the clinicians. Clinical significance: The stability of the varnish plays a vital role in maintenance of its long-term efficacy. The chemical nature along with the ability of the material to interact with the substrate plays a major role in management of DH.