Background: Oseltamivir, a neuraminidase inhibitor (NAI), is the primary and first-line anti-influenza drug. In recent years, more and more oseltamivir resistant strains appeared frequently. Purpose: To identify anti-influenza candidates to overcome oseltamivir resistance from traditional Chinese medicine. Methods: High-throughput screening platform was used to screen candidates with oseltamivir sensitive and resistant neuraminidase (NA) inhibitory activities from traditional Chinese medicine (TCM) and in validation of potential active components in vitro. Molecular docking is used for virtual screening of TCM compound libraries and analysis of the molecular mechanism of active compounds. Results: Six out of 188 TCM formula granules were screened out with good inhibitory activities in oseltamivir sensitive (H5N1) and resistant NA (H274Y mutant). A compound library containing 679 candidates in the above six plants were docked into binding pockets of H5N1 NA and its H274Y mutant. The results indicated that diverse structural types, including flavonoids glycosides, alkaloids, caffeoylquinic acid derivatives, etc. showed good docking scores on the two NAs. Furthermore, 10 representative compounds tested in vitro showed inhibitory activities in both oseltamivir sensitive and resistant NA. The docking simulations revealed that natural TCM molecules bind with NA in an absolute different mode compared with oseltamivir, which occupy the extra cavities adjacent to the active site of NA, thus might make them non-susceptible to be influenced by mutation around the active sites. Conclusions: The combination of high-throughput screening and molecular docking can efficiently screen out candidates that have the potential to overcome oseltamivir resistance from TCM. © 2023