Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titer of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.