Aim: This study aimed to evaluate the bacterial DNA and RNA levels within the access ‎cavity before and after disinfection.   ‎ Materials and methods: Twenty-seven patients with necrotic teeth were included. Strict aseptic measures were ‎applied, including plaque removal, disinfection of teeth, and application of rubber dam, ‎using 30% hydrogen peroxide (H2O2) followed by 2.5% sodium hypochlorite (NaOCl). ‎NaOCl activity was neutralised using 10% sodium thiosulfate with saline as the final rinse. ‎After access cavity preparation, bacterial DNA and RNA samples were collected using ‎sterile paper points, before and after using the same disinfection protocol. Samples were ‎verified by Real-time polymerase chain reaction using universal 16S rRNA primers. Data ‎were analysed using the Wilcoxon matched pairs and Fisher's exact tests. The significance ‎level was set at P<0.05. ‎ Results: Bacterial DNA and RNA levels were reduced following a disinfection protocol (P<0.0001). ‎Bacterial DNA was detected in all the pre- and post-disinfection samples. There was a ‎significant decrease in the proportion of patients with detectable RNA levels after ‎disinfection (P<0.0001). ‎ Conclusion: Access cavity disinfection before root canal sampling is crucial to avoid bacterial ‎infiltration into the root canal.‎