Background Effective disinfection of the root canal system is critical to the success of endodontic treatment. Various irrigation protocols are employed to eliminate microbial presence, yet the efficacy of these protocols remains a topic of ongoing research. This study aims to evaluate and compare the microbiological effectiveness of different irrigation protocols in disinfecting the root canal system. Materials and Methods This original study was conducted on 120 extracted human teeth with single canals, randomly assigned into four groups of 30 samples each. The groups were treated using different irrigation protocols: Group A (5.25% Sodium Hypochlorite), Group B (2% Chlorhexidine), Group C (EDTA followed by 5.25% Sodium Hypochlorite), and Group D (MTAD). Pre- and post-irrigation microbial samples were collected using sterile paper points, cultured on specific media, and incubated for 48 hours at 37°C. The reduction in microbial colony-forming units (CFUs) was measured and compared across groups. Results The mean CFU reduction was highest in Group D (MTAD) with a 98.2% reduction, followed by Group C (EDTA + NaOCl) with a 96.5% reduction. Group A (NaOCl) showed an 85.4% reduction, while Group B (Chlorhexidine) demonstrated the least efficacy with a 78.9% reduction. Statistical analysis using ANOVA indicated a significant difference in microbial reduction among the groups (p < 0.05).