Through evolution, Hepatitis B Virus (HBV) developed highly intricate mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. Yet a clear picture of viral hitchhiking of cellular processes with spatial resolution is still largely unsolved. Here, by leveraging bDNA-based fluorescence in situ hybridization (FISH) combined with immunofluorescence, we developed a microscopic approach for multiplex detection of viral nucleic acids and proteins, which enabled us to probe some of the key aspects of HBV life cycle. We confirmed the slow kinetics and revealed the high variability of viral replication at single-cell level. We directly visualized HBV minichromosome in contact with acetylated histone 3 and RNA polymerase II and observed HBV-induced degradation of Smc5/6 complex only in primary hepatocytes. We quantified the frequency of HBV pregenomic RNAs occupied by translating ribosome or capsids. Statistics at molecular level suggested a rapid translation phase followed by a slow encapsidation and maturation phase. Finally, the roles of microtubules (MTs) on nucleocapsid assembly and virion morphogenesis were analyzed. Disruption of MTs resulted in the perinuclear retention of nucleocapsid. Meanwhile, large multivesicular body (MVB) formation was significantly disturbed as evidenced by the increase in number and decrease in volume of CD63+ vesicles, thus inhibiting mature virion secretion. In conclusion, these data provided spatially resolved molecular snapshots in the context of specific subcellular activities. The heterogeneity observed at single-cell level afforded valuable molecular insights which are otherwise unavailable from bulk measurements.