Considering the need for discovery of new antiviral drugs, in view to combat the issue of resistance particularly to anti-influenza drugs, a series of 2´-amino, 3´-amino and 2´, 4´-dihydroxy chalcone derivatives were designed. Structure-based drug design was used to design inhibitors of influenza virus - H1N1 neuraminidase enzyme. These were further optimized by a combination of iterative medicinal chemistry principles and molecular docking. Based on the best docking scores, some chalcone derivatives were synthesized and characterized by infrared spectroscopy (IR) and proton nuclear magnetic resonance (NMR). The molecules were evaluated for their anti-influenza action against influenza A/Pune isolate/2009 (H1N1) virus by in vitro enzyme-based assay (neuraminidase inhibition assay). We have then selected few of them for multinuclear NMR studies, 31P NMR, in order to probe the molecular mechanism of their antiviral action. Reasonably good correlation between docking scores; anti-influenza activity; and 31P NMR results were observed. The computational predictions were in consensus with the experimental results. It was observed that among tested compounds, derivative 1A, viz. 2´, 4´-dihydroxy-4-methoxy chalcone, showed highest activity (IC50 = 2.23 μmol/l) against the virus under study. This derivative 1A can be explored further to provide a future therapeutic option for the treatment and prophylaxis of H1N1 viral infections.