The development of organic molecules to target nucleic acid is an active area of research at the interface of chemistry and biochemistry, which involves DNA binding, nuclear imaging, and antitumor studies. These molecules bind with DNA through covalent interactions, electrostatic interactions, or intercalation. However, they are less permeable to membrane, and they have a significant cytotoxicity, which limits their application under in vivo conditions. In the present work, various mono- and disubstituted 1,8-naphthalimides-based derivatives (S-12, S-13, S-15, and S-21) have been synthesized and characterized through various spectroscopic techniques. Among these, 3-amino-4-bromo-1,8-naphthalimide (S-15) was found to have an attractive water solubility and act as a nuclear imaging agent. The spectroscopic absorption and emission data showed that S-15 has a strong affinity for salmon sperm DNA with a binding constant of 6.61 × 104 M–1, and the ratiometric fluorescence intensity (I489/I552) of S-15 has a linear relationship in the 0–50 μM range of DNA concentrations. It intercalates with DNA through the hydrophobic planar naphthalimide core as confirmed through cyclic voltammetry, circular dichroism, 1H NMR titration, and thermal denaturation studies. Positively charged amine groups also participate in H-bonding with the bases and backbone of DNA. The S-15 intercalator showed a large Stokes shift and photostability, which made it attractive for direct imaging of Legionella pneumophila, without the need for a prior membrane permeabilization.