The effect of substituents on biological activity of Schiff bases was investigated using Schiff bases derived from the condensation of aniline with salicyaldehyde, 5-methoxysalicyaldehyde, 5-nitrosalicyladehyde and 2- hydroxynaphtaldehyde. The electronic absorption spectra of the compounds in DMF and ethanol reveal that compounds containing the electron withdrawing nitro or naphtyl groups exist in the keto-amine form in both solvents. The compounds were screened against some clinically important bacteria namely Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 13315), Enterococcus feacalis (ATCC 29212) and Staphylococcus aureus (ATCC25923) using the agar ditch method and results show that 5-methoxysalicyaldehyde Schiff base exhibited highest activity.