The novel 1-(2-fluorovinyl)-4-quinolone-3-carboxylic acid derivatives Z-15a-c, E-15a-c, Z-16a-c, and E-16a-c, conformationally restricted analogues of fleroxacin (5), were synthesized, and their in vitro antibacterial activity was evaluated. A dehydrosulfenylation of a 2-fluoro-2-[(4-methoxyphenyl)sulfinyl]ethyl group was employed as a key step for the construction of a 2-fluorovinyl group at the N-1 position. It appeared evident that the Z-isomers Z-15a-c and Z-16a-c exhibited 2- to 32-fold more potent in vitro antibacterial activity than the corresponding E-isomers E-15a-c and E-16a-c. Furthermore, since Z-15b showed in vitro antibacterial activity and DNA gyrase inhibition comparable to that of 5, it was hypothesized that the conformation of Z-15b would be equivalent to the active conformer of 5. The results revealed that the antibacterial Z-1-(2-fluorovinyl)quinolone derivatives carry the novel N-1 substituent of the fluoroquinolones.