In this study, ZnFe2O4 and calcium-substituted Zn1-xCaxFe2O4 (x = 0.2, Ca2-ZF & x = 0.4, Ca4-ZF) are synthesized using Morinda citrifolia L. leaves extract via the microwave method, and calcined at 800 °C for 4 h to enhance the crystallinity. The purity and structural features of the ferrites were analyzed using powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR) techniques confirming single phase formation and functional groups. Morphology and elemental presence were examined using high-resolution scanning electron microscopy (HR-SEM) and energy dispersive X-ray analysis (EDX) that exhibit polyhedral morphology of the particles and confirm elemental composition. The bandgap of Zn1-xCaxFe2O4 determined using the Kubelka method from UV-DRS data, is found to increase as calcium concentration and is in the range 1.73–1.91 eV. Room-temperature VSM studies showed a transition from superparamagnetic behavior to soft ferromagnetic behavior upon Ca substitution. Zn0.6Ca0.4Fe2O4 (Ca4-ZF) composition exhibits maximum saturation magnetization, remnant magnetization and coercivity of 196 x10−3emu/g, 0.411 x10−3emu/g, and 0.091 Oe, respectively, making it suitable for biomedical applications such as cell separation, detection, and gene delivery support. The antibacterial and antifungal studies demonstrate better inhibition for ZF than for Cax-ZF compounds. The pure ZF shows a maximum zone of inhibition against Bacillus subtitles bacteria and Candida albicans fungus of 23.7 mm/250 μg/mL and 28.1 mm/250 μg/mL, respectively.