The gradual Cl replacement reactions of NN (1-3) or NO spirocyclic monoferrocenyl cyclotriphosphazenes (4 and 5) with the potassium salt of 4-hydroxy-3-methoxybenzaldehyde (potassium vanillinate) resulted in the mono (1a-5a), geminal (gem-1b-5b), non-geminal (cis-5b and trans -1b-4b), tri (1c, 3c-5c) and tetra-vanillinato-substituted phosphazenes (1d-5d). All the phosphazene derivatives have stereogenic P-center(s), except tetra-substituted ones. The vanillinatophosphazenes have reversible voltammograms with one-electron anodic and cathodic peaks which are attributed to ferrocenyl redox probe. The structures of the new phosphazene compounds were determined by FTIR, MS, (1)H, (13)C{(1)H} and (31)P{(1)H} NMR spectral data. The solid-state structure of cis -5b was examined by single-crystal X-ray diffraction techniques. In addition, the compounds were tested in HeLa cancer cell lines using MTT assay. The 12 phosphazene derivatives were screened for antimicrobial activity, and 3c was very effective against S. aureus even at 4.88 µM concentration, taking into account the MIC values. Besides, interactions between the phosphazenes and pBR322 plasmid DNA were also investigated.