Covalently closed circular DNA (cccDNA) is a stable episomal form of the hepatitis B virus (HBV) genome that serves as the template for viral transcription and replication and represents a major barrier to HBV cure. Here, we investigated chromatin accessibility patterns of cccDNA in HBV-infected hepatocyte cells at single-molecule resolution. We found that most cccDNA copies exhibited limited accessibility around nucleotides 800–1000, a region overlapping the polymerase open reading frame and the pregenomic RNA transcriptional region. Notably, a small subset of cccDNA showed detectable accessibility at this site, suggesting the presence of heterogeneous chromatin states. Based on this observation, we targeted this accessibility-associated region using a CRISPR/Cas9-based approach and observed reductions in HBV DNA-related signals, including cccDNA-enriched fractions and total HBV DNA levels across complementary experimental systems. These findings suggest that chromatin accessibility profiling may provide an additional framework for identifying candidate cccDNA target regions. Our study provides a proof-of-concept for accessibility-informed HBV targeting and supports further investigation of chromatin-associated vulnerability within HBV cccDNA.