The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an antiviral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding, a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, druglike compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nanomolar affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays.