In the present work, N-[2-(thienyl)-2-(chlorobenzyloxyimino)ethyl]enoxacin derivatives (4a-f) were synthesis by the reaction of enoxacin with 2-bromo-1-(thiophen) ethanone O-2-chlorobenzyl oxime derivatives (3a-f) in DMF in the presence of NaHCO3 at room temperature. The synthesized compounds were tested in vitro on human tumor cell lines. Preliminary screening showed 4b and 4e demonstrated significant growth inhibitory potential against all evaluated cell lines. The results of structure-activity relationship study exhibited that quinolone derivatives containing 3-chlorobenzyloxime are superior in cytotoxic potential compared to 2 and 4-chloro derivatives.