Eighteen novel levofloxacin-thiadiazole HDACi conjugates were designed and synthesized from levofloxacin. The chemical structures of all conjugates were confirmed by ¹H NMR, ¹³C NMR and HR-MS spectra. The inhibitory activities of new conjugates were evaluated in an assay with a HDACs reagent kit, and their anti-tumor activities were tested in CCK-8 assay. The results showed that these new conjugates displayed potent inhibitory activity against HDACs, and the hydroxamate conjugates exhibited more potent activity than carboxylic acid and benzamide derivatives. Specifically, conjugate 5d exhibited the most potent anti-HDAC1 (IC₅₀=0.031±0.011 μmol·L^-1) and HDAC6 (IC₅₀=0.019±0.006 μmol·L^-1) activities, which was more potent than SAHA. Molecular docking studies suggest that the hydroxamate group of conjugate 5d was deeply inserted into the active site to interact with the residues in coordination with the zinc ion. Additionally, the thiadiazole group of conjugate 5d also engaged in hydrogen bonding with F679 in HDAC6, which had been linked to the selectivity of the HDAC isoforms. Moreover, these conjugates displayed significant antiproliferative effects on SW620, MGC-803, PC-3, NCIH460, MCF-7 and HepG2 cells, in particular, conjugate 5d showed the greatest potency against MGC-803 (IC₅₀=0.7±0.05 μmol·L^-1), NCIH460 (IC₅₀=2.3±0.421 μmol·L^-1), MCF-7 (IC₅₀=1.6±0.56 μmol·L^-1) and HepG2 (IC₅₀=3.9±0.26 μmol·L^-1), which was >3-fold more potent than SAHA. Additionally, all conjugates were nontoxic to health GES-1 cells, while SAHA showed some toxicity.