Fluoroquinolones (FQs) are well known antibacterial chemotherapeutics with DNA gyrase inhibitory activity and a potential urease inhibitory activity. New FQ hybrids (4a−h and 5a−h) have been synthesized by combining ciprofloxacin or norfloxacin with different thiouracil and thioxopyrimidine derivatives. The purity of the newly developed compounds was assessed through thin-layer chromatography, and the elucidation of their structures was confirmed through proton nuclear magnetic resonance, infrared spectroscopy, high resolution mass spectrometry, and microanalyses. The new hybrids were evaluated for their antibacterial, DNA gyrase, and urease inhibitory activities. The potential co- and cross-resistance in Escherichia coli (E. coli) as well as the frequencies of the resistance were evaluated for the new hybrids. The new hybrid compounds displayed a significant antibacterial activity against a panel of Gram-positive and Gram-negative bacterial strains. The majority of the tested ciprofloxacin and norfloxacin hybrids displayed a superior activity against E. coli. The 4 g and 4 h hybrids were the most active E. coli DNA gyrase inhibitors (IC₅₀: 0.62 and 0.58 µM, respectively), and displayed an inhibitory activity that was comparable to that of ciprofloxacin (IC₅₀: 0.40 µM). The 5 g and 5e hybrids exhibited an improved inhibitory activity (IC₅₀: 0.76 and 0.80 µM, respectively) when compared to norfloxacin (IC₅₀: 0.93 µM). Most of the tested hybrids were shown to be able to additionally inhibit the urease activity (IC₅₀ range: 9.71–80.64 μM). Hybrids 4e and 5c were the most potent urease inhibitors (IC₅₀: 11.68 and 9.71 μM, respectively), capable of displaying a 3 − 4-fold increase in their activity when compared to ciprofloxacin, norfloxacin, and thiourea (IC₅₀: 42.74, 36.07, and 32.25 μM, respectively).