A highly sensitive cell-based imaging assay has been used to screen a 200,000 compounds library for potential influenza antivirals. Compounds were screened at a concentration of 10 μM against the influenza strain A/Udorn/72 (H3N2) and the duplex capability of the assay was taken advantage of to select compounds with no or low cytotoxicity. The assay was also used for confirmation in concentration-response of the active compounds. In the set of confirmed hits, three major structural clusters, 23 minor clusters and 86 singletons were identified. Further evaluation of anti-influenza activity was performed for two additional influenza strains of the H1N1 subtype, A/WSN/33 and A/California/07/09. Three compounds from cluster A, SRI-44211, SRI-44215 and SRI-44221 showed selectivity indices for the pandemic strain A/California/07/09 in the range of 54.3 to 252, providing evidence that the core structure of cluster A could play a relevant role in their inhibitory potency and may serve as a starting point for future hit-to-lead optimization efforts.