During the screening program for anti-influenza agents from medicinal plants, the ethanolic extract of Cleistocalyx operculatus leaves was found to exhibit potential neuraminidase (NA) inhibitory activity. Bioassaydirected fractionation led to the isolation of two new acetophenones (1 and 2) and one new flavanone (3), along with six known compounds (4–9). The structures of all isolated compounds were elucidated using various spectroscopic methods and through comparison with the previous literature. Compounds 6 and 8 exhibited strong enzymatic inhibition on various neuraminidases from different influenza viruses, including H1N1, H9N2, novel H1N1, and oseltamivir-resistant novel H1N1 (H274Y mutation) expressed in HEK293 cells (IC50 values ranging from 5.07 ± 0.94 μM to 9.34 ± 2.52 μM, respectively). Kinetic experiments revealed the non-competitive inhibitory mode of both compounds 6 and 8. Furthermore, these flavonoids reduced the cytopathic effect of the H1N1 virus in MDCK cells. The present study suggests the potential of two flavonoids (6 and 8) as new lead compounds for the development of novel NA inhibitors in the future.