Twelve spiro thiazolidinone compounds (
A–L
) were synthesized via either conventional thermal or ultrasonication techniques using Fe
2
O
3
nanoparticles. The modification of the traditional procedure by using Fe
2
O
3
nanoparticles led to enhancement of the yield of the desired candidates to 78–93% in approximately half reaction time compared with 58–79% without catalyst. The products were fully characterized using different analytical and spectroscopic techniques. The structure of the two derivatives 4-phenyl-1-thia-4-azaspirodecan-3-one (
A
) and 4-(p-tolyl)-1-thia-4-azaspirodecan-3-one (
B
) were also determined using single crystal X-ray diffraction and Hirshfeld surface analysis. The two compounds (
A
and
B
) were crystallized in the orthorhombic system with
Pbca
and
P2
1
2
1
2
1
space groups, respectively. In addition, the crystal packing of compounds revealed the formation of supramolecular array with a net of intermolecular hydrogen bonding interactions. The energy optimized geometries of some selected derivatives were performed by density functional theory (DFT/B3LYP). The reactivity descriptors were also calculated and correlated with their biological properties. All the reported compounds were screened for antimicrobial inhibitions. The two derivatives,
F
and
J
, exhibited the highest levels of bacterial inhibition with an inhibition zone of 10–17 mm. Also, the two derivatives,
F
and
J
, displayed the most potent fungal inhibition with an inhibition zone of 15–23 mm. Molecular docking investigations of some selected derivatives were performed using a B-DNA (PDB: 1BNA) as a macromolecular target. Structure and activity relationship of the reported compounds were correlated with the data of antimicrobial activities and the computed reactivity parameters.