In this study, magnetite nanoparticles were successfully synthesized using Calotropis procera aqueous leaf extract. The effect of the whole extract and some of its phytochemicals on the particle size distribution was determined using Dynamic light scattering (DLS) while other characterizations such as UV–vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy and Scanning electron microscopy (SEM) were carried out. The antimicrobial activity against selected microorganisms was also studied using the agar well diffusion method. The leaf extract showed the presence of phenols, flavonoids, saponins, alkaloids and tannins. The magnetite nanoparticle synthesized using the leaf extract (Calotropis procera/Fe3O4) had an average particle size of 11.1nm with a PDI of 0.142 while the phenolic, flavonoid and saponin extracts of Calotropis procera leafs produced magnetites with average particle sizes of 62.83nm, 68.02nm and 134nm respectively. The UV-vis spectra displayed the characteristic plasmon resonance peak at 420nm, the FTIR plots highlighted important functional groups including peaks between 600cm-1 and 400cm-1 confirming the formation of iron oxide while the SEM micrographs showed the nanoparticles were mainly irregular in shape with areas of agglomeration. Calotropis procera/Fe3O4 displayed significant anti-microbial activity against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, Aspergillus niger and Fusarium oxysporum while no activity was recorded against Escherichia coli. The study indicated that Calotropis procera leaf extract was suitable for the green synthesis of magnetite with properties that suggest that it could be potentially applied as antimicrobial nanofillers.