A series of novel fluoroquinolone-Safirinium dye hybrids was synthesized by means of tandem Mannich-electrophilic amination reactions from profluorophoric isoxazolones and antibiotics bearing a secondary amino group at position 7 of the quinoline ring. The obtained fluorescent spiro fused conjugates incorporating quaternary nitrogen atoms were characterized by <sup>1</sup>H NMR, IR, MS, and elemental analysis. All the synthetic analogues (3a-h and 4a-h) were evaluated for their in vitro antimicrobial, bactericidal, and antibiofilm activities against a panel of Gram positive and Gram-negative pathogenic bacteria. The most active Safirinium Q derivatives of lomefloxacin (4d) and ciprofloxacin (4e) exhibited molar-based antibacterial activities comparable to the unmodified drugs and displayed considerable inhibitory potencies in E. coli DNA gyrase supercoiling assays with IC<sub>50</sub> values in the low micromolar range. Zwiterionic hybrids were noticeably less lipophilic than the parent quinolones in micellar electrokinetic chromatography (MECK) experiments. The tests performed in the presence of phenylalanine-arginine β-naphthylamide (PAβN) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) revealed that the conjugates are to some extent subject to bacterial efflux and cellular accumulation, respectively. Moreover, the hybrids did not exhibit notable cytotoxicity towards the HEK 293 control cell line and demonstrated low propensity for resistance development, as exemplified for compounds 3g and 4b. Finally, molecular docking experiments revealed that the synthesized compounds were able to bind in the fluoroquinolone-binding mode at S. aureus DNA gyrase and S. pneumoniae topoisomerase IV active sites.