6-Fluoroquinolones are useful antimicrobial agents against gram-positive and gram-negative bacteria and some mycobacterial species as well. Although the diffusion through porins in gram-negative bacteria is well established, other mechanisms such as the hydrophobic pathway through the apolar regions of the bilayer and the self-promoted pathway appear to be relevant or concomitant with the hydrophilic pathway in many cases. This article discusses the interaction of ciprofloxacin (CPX) and two new synthesized compounds (M3CPX and M4CPX)-with a methyl group attached at the N3 and N4 positions of the piperazynil ring of the CPX-with liposomes and supported planar bilayers (SPBs) of Escherichia coli. Binding experiments using ANS revealed that the three compounds interact electrostatically with the bilayer. The variations in the electrostatic surface potential, which is always positive, were higher for M3CPX than for CPX or M4CPX. Related to that, the SPBs of E. coli were more affected by M3CPX than by the other two compounds, as judged by the analysis of the atomic force microcopy (AFM) images. The in situ injection of the three 6-fluoroquinolones (6-FQs) induced different changes in height, roughness (Ra), and area covered by the SPBs.