Simian virus 40 large T-antigen (SV40 LT-Ag) is a 708 amino acid nuclear phosphoprotein. Among many functions of LT-Ag is its ability to perform as an ATPase-helicase, catalyzing the unwinding of viral genome during replication. The LT-Ag has been employed in the studies of helicase structure and function, and has served as a model helicase for the screening of antiviral drugs that target viral helicase. In this study, using in vitro enzyme assays and in silico computer modeling, we screened a batch of 18 fluoroquinolones to assess their potential as antivirals by virtue of their inhibition of the LT-Ag helicase. We found all fluoroquinolones to be inhibitory to the helicase activity of LT-Ag. In our docking analysis, most of these tested drugs showed similarity in their interactions with LT-Ag. Our study shows the potential of fluoroquinolones as antiviral drugs and of SV40 LT-Ag as a model protein for screening drugs against viral helicases.