In this study, primarily, tetrachloro-2-pyridylmethylspiro-N/N-ethylenediaminocyclotriphosphazenes (4 and 5) were prepared from the reactions of equimolar amounts of N-alkyl-N 0 -2-pyridylmethylethylenediamines, [PyCH 2 NH(CH 2 ) 2 NHR (R:CH 3 , 2 and R:C 2 H 5 , 3)], and hexachlorocyclotriphosphazene [(HCCP, trimer), N 3 P 3 Cl 6 (1)]. Compounds 4 and 5 react with secondary amines to yield the fully substituted tetraamino-2-pyridylmethylspiro-N/N-ethylenediaminocyclotriphosphazenes (4a-5c) in dry THF, respectively. These derivatives were obtained by both conventional and microwave-assisted reactions, and their yields were compared. In addition, it should also be noted that these cyclotriphosphazenes were obtained for use in the investigation of their spectral, crystallographic, antimicrobial, and anticancer properties. The structures of the compounds were elucidated by spectral data. The crystal structures of 4, 5a, and 5c were clarified by the single crystal X-ray diffraction technique. Antibacterial activities and biofilm preventions of 4a-c and 5a-c against bacteria, and their antifungal activities against fungi were investigated. According to broth microdilution method, the results showed that all compounds were highly effective against C. albicans. The interactions of these compounds with DNA and their cytotoxic activities against L929 fibroblast and A549 lung cancer cell lines were investigated. In addition, Density Functional Theory (DFT) calculations of 4, 5a, and 5c were made, and molecular docking studies with DNA and cytochrome P450 enzyme were presented.