Background Toothbrushes are the most commonly used mechanical plaque control methods, and they are said to harbor microorganisms if not stored properly following usage. Objective An assessment of the influence of a decontaminating agent (sterile water (control)/chlorhexidine mouthwash/herbal mouthwash) on the properties of toothbrush bristles following storage for 24 hrs by means of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Methodology The study involves a total of 24 slim soft toothbrushes (six samples per group) with different decontaminating agents: Group 1: Vented container, Group 2: Sterile water, Group 3: 0.2% Chlorhexidine mouthwash, and Group 4: Herbal mouthwash. The toothbrushes were stored in their respective containers for 24 hours, following which, snippets of toothbrush bristles were collected and tested for the evaluation of wear using SEM and the biochemical alteration occurring in the bristles was assessed using FTIR. Results and conclusion The Group 2 and Group 3 samples showed significant wear (Score 2) under SEM evaluation, and FTIR spectroscopy evaluation revealed that changes occur in the fingerprint region of infrared spectroscopy owing to chemical bond alteration following decontamination. Considering the benefits we acquire in terms of microbial load reduction, decontamination is recommended, though it might necessitate frequent changing of toothbrushes.