A series of 9 monocationic styryl dyes was prepared in moderate to good yields via Knoevenagel condensation between various CH active N-quaternary heterocycles and 4-(4-methylpiperazin-1-yl)benzaldehyde. Structural elucidation of the newly synthesized fluorophores was achieved by H-1 NMR, C-13 NMR, Se-77-NMR and high-resolution mass spectrometry (HRMS) in ESI mode. Novel dyes are characterised by very pronounced Stokes shift (94-203 nm), while fluorescence quantum yields (0.18-2.38 x 10(-2)) were strongly dependent on the selection of N-quaternary heterocycle. New dyes bind to DNA/RNA by micromolar affinity, yielding strong fluorescence increase. Among the studied series the strongest enhancement was observed for the two quinolinium derivatives and the benzo[e]indole analogue. The dye emission response selectivity between ds-DNA and ds-RNA was opposite for para-quinolinium and ortho-quinolinium analogue, stressing the importance of fine tuning of fluorophore by means of electronic properties and positioning within the DNA/RNA binding site. Combined results of several methods support binding of dyes within DNA minor or RNA major groove, with exception of indole and benzo[e]indole analogues, which show partial intercalation. Majority of dyes investigated in this work showed negligible cytotoxic activity, with an exception of the benzo[e]indole-dye micromolar cytotoxicity. The majority of the studied dyes exhibit localization within mitochondria, with two exceptions. The benzo[d] [1,3] selenazole-derived dye preferentially localizes in lysosomes, while benzo[e]indole-dye is equally distributed between mitochondria and lysosomes at variance to indole-dye highly selective for mitochondria, suggesting different intracellular mechanisms of these two related dyes. Observed bioactivity of benzo[e]indole-dye combined with easily monitored localization by strong fluorescence makes this dye potential theranostic agent.