We report herein synthesis, characterization and antimicrobial activity of four Shiff bases derived from hexamethylenediamine. Substitution of a nitro group on each aromatic ring in ortho, meta or para positions of N,N'-bis(phenylmethyl)hexane-1,6-diimineallowed to have a homogeneous series of positional isomers. These four symmetric diimine Schiff bases were characterized by conventional spectrometry methods (NMR, IR, MS), then tested against Gram-positive and Gram-negative bacterial strains. Among them, compounds 1b, 1c, 1d were found to be active against bacterial strain Staphylococcus aureus CIP with MIC value of 375μg/ ml, 187.5μg /ml and 375μg /ml respectively. Candida Albicans fungal strain showed resistance to all synthesized Schiff base compounds, butin the other hand, Candida glabrata has been sensitive to all compounds with MIC of 1500μg/ ml and one more time except 1a.