It was reported recently that a new aryl methyldiene rhodanine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.