The Cl substitution reactions of the N/N (1-3) and N/O (4 and 5) spirocyclic monoferrocenylphosphazenes with 1,4-dioxa-8-azaspiro[4,5]decane (DASD) produce the mono- (1a-5a), geminal- (1b-5b) and tetrakis-DASD-substituted ferrocenylspirocyclotriphosphazenes (1c-5c). The mono- and geminal-DASD-substituted phosphazenes have two and one stereogenic P-centers, respectively. The structures of the compounds were established by spectroscopic techniques. The molecular structures of 3a and 2b were evaluated using X-ray crystallography. Additionally, the ultrathin and highly ordered Langmuir-Blodgett (LB) films of 3a and 2b were also prepared. The characterizations of the LB films were done using p-polarized grazing angle (GAIR) and horizontal attenuated total reflectance (HATR) techniques. On the other hand, the antimicrobial activities of the eight phosphazene derivatives against G(+) and G(-) bacteria and fungi were investigated. Furthermore, the interactions between the compounds and plasmid DNA were studied by agarose gel electrophoresis.