Huang-Lian-Jie-Du-Tang (HLJDT), a traditional formula with four TCM herbs, has been used for hundred years for different diseases. The current study aimed to assess the inhibitory activity of HLJDT against H1N1 neuraminidase (NA-1), and identify potent NA-1 inhibitors from its plasma profile. The in vitro NA-1 study has shown that the water extract of HLJDT potently inhibited NA-1 (IC50 = 112.6 μg/ml; Ki = 55.6 μg/ml) in a competitive mode. The IC50 values of the water extracts of its four herbs were as follows: Coptidis Rhizoma, 96.1 μg/ml; Phellodendri Chinensis Cortex, 108.6 μg/ml; Scutellariae Radix, 303.5 μg/ml; Gardeniae Fructus, 285.0 μg/ml. Thirteen compounds found in the plasma profile of HLJDT were also identified as potent NA-1 inhibitors, which included jatrorrhizine, palmatine, epiberberine, geniposide, oroxylin A, berberine, coptisine, baicalein, wogonoside, phellodendrine, wogonin, oroxylin A-7-O-glucuronide and baicalin (sorted in ascending order by their IC50 values). Their inhibitory activities were consistent with molecular docking analysis when considering crystallographic water molecules in the ligand-binding pocket of NA-1. Our current findings suggested that HLJDT can be used as a complementary medicine for H1N1 infection and its potent active compounds can be developed as NA-1 inhibitors.