Yellow fever virus (YFV), a mosquito-borne pathogen responsible for acute haemorrhagic fever, remains a significant health threat due to recurrent outbreaks, high disease burden, vaccine shortages, and the lack of antiviral treatments. Here, we report the identification of YFV inhibitors targeting the NS4B protein. Among a series of heterocyclic compounds containing hexahydro-2H-4,6-(epoxymethano)chromene moieties, compound I7-20-1 exhibited the potent antiviral activity in Huh7 cells (EC₅₀ = 3.4 µM, CC₅₀ = 60 µM) resulting in a ~3 log reduction in viral titres. No antiviral activity was observed against other flaviviruses, alphaviruses, or enteroviruses, indicating YFV-specific activity. Time-of-addition experiments showed that I7-20-1 was most effective when added up to 4 hours post-infection, indicating that it acts during an early stage of the viral replication cycle. Resistance selection identified a I84T substitution in the NS4B viral protein, located within transmembrane domain 2. Reverse engineering confirmed the resistant phenotype (>9-fold resistance). Notably, cross-resistance studies with previously described YFV NS4B inhibitors, showed that the I84T mutant showed cross-resistance to CCG-4088, but not to BDAA. Molecular dynamics analysis applied to AlphaFold-generated proteins suggests that the I84T mutation affects NS4B structural conformation, reducing ligand-binding specificity compared to wild type. Ongoing studies aim to elucidate the molecular mechanism of action, including whether viral RNA replication is directly affected, and to evaluate the activity of I7-20-1 in YFV-infected human liver organoids. In summary, we identified a new class of YFV inhibitors targeting a novel NS4B binding site, with a resistance profile distinct from the BDAA inhibitor.