5-(Ethylamino)-9-diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a photosensitizer (PS) with broad antimicrobial photodynamic activity. The objective of this study was to determine the antimicrobial photodynamic effect of side chain/end group modifications of EtNBS on two representative bacterial Gram-type-specific strains. Two EtNBS derivatives were synthesized, each functionalized with a different side-chain end-group, alcohol or carboxylic acid. In solution, both exhibited photochemical properties consistent with those of the EtNBS parent molecule. In vitro photodynamic therapy experiments revealed an initial Gram-type-specificity with two representative strains; both derivatives were phototoxic to Staphylococcus aureus 29,213 but the carboxylic acid derivative was nontoxic to Escherichia coli 25,922. This difference in photodynamic efficacy was not due to a difference in the binding of the two molecules to the bacteria as the amount of both derivatives bound by bacteria was identical. Interestingly, the carboxylic acid derivative produced no fluorescence emission when observed in cultures of E. coli via fluorescence microscopy. These early findings suggest that the addition of small functional groups could achieve Gram-type-specific phototoxicity through altering the photodynamic activity of PSs and deserve further exploration in a larger number of representative strains of each Gram type.