Accumulated clinical studies have demonstrated that histone deacetylase (HDAC) inhibitors show great potential for the treatment of cancer. SAHA (Vorinostat, trade name Zolinza) was approved by the FDA in 2006 for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma. As a continuity of our ongoing effort to identify novel small molecules targeting these important enzymes, we designed and synthesized two series of isatin-3'-oxime- and isatin-3'-methoxime-based hydroxamic acids (3a-g and 6a-g) as analogues of SAHA. Generally in both series it was found that, compounds bearing no substituent or with 5'-F, 5'-Cl, 7'-Cl substitutents on the isatin moiety exhibited good inhibition against histone-H3 and histone-H4 deacetylation at the concentrations of 1 μM, as evaluated by Western Blot assay. The compounds also displayed potent cytotoxicity against five cancer cell lines with IC50 values of as low as 0.08 μM, more than 45-fold lower than that of SAHA. Docking study performed with selected compounds 3a and 6a revealed that these compounds bound to HDAC8 with higher affinities compared to SAHA. Compounds 3a and 6a also bound to HDAC2 at the binding site with high binding affinity. These findings should encourage further elaboration with the isatin moiety to produce more potent HDAC inhibitors with potential anticancer activity.