To study the influence of the substituent at the N-10 position on antiviral activity, the chiral quaternary ammonium salt derivatives of R- and S-tylophorine were designed, synthesized, and evaluated for antiviral activity against tobacco mosaic virus (TMV). The bioassay results indicated that most of the designed structural analogues showed good in vivo anti-TMV activity, among which propargyl quaternary ammonium salt compound S-7b showed the best anti-TMV activities (80.5%, 77.6%, 76.6%, 82.1%) at 500 μg/mL both in vitro and in vivo in the laboratory. In the field trials of antiviral efficacy against TMV, S-7b as well exhibited better activities than control plant virus inhibitors. The stability of compound S-7b was obviously increased, and its solubility was more than 500-times higher than that of S-tylophorine. Therefore, chiral quaternary ammonium salt S-7b was expected to be developed as a promising candidate as an inhibitor of plant virus.