Fly ash (FA) is produced from coal power plants’ combustion. FA is used in the concrete industry, as an ingredient in the brick and paving. Knowledge of the chemical composition and toxic metal content in FA is essential for evaluating its environmental risks. This study aimed to assess FA purification effect on its antibacterial activity against Escherichia coli and Bacillus cereus, by calculating percent bacterial reduction. Moreover, centrifugation time effect on the purification process was evaluated. Chemical composition and properties of purified FA were determined and compared with raw FA, using Fourier transform Infrared (FTIR); X-ray diffraction (XRD); X-ray photoelectron spectroscopy (XPS); energy-dispersive X-ray (EDXA); carbon, hydrogen, nitrogen, and sulfur (CHNS) elemental analysis; moisture content; and loss-of-ignition. Particle size was predicted by using dynamic laser scattering, BET and scanning electron microscopy (SEM). The CHNS results showed that purified FA contains the highest carbon content (88.9%), as compared to raw FA (82.1%). The particle size distribution (PSD) of FA microspheres ranges from 48.53 ± 17.9 to 52.98 ± 19.4 µm by using SEM. PSD, using dynamic laser scattering, showed polydispersed and non-uniform size in raw FA, ranging in size from 107.1 to 1027 nm, while purified FA manifests a monodispersed size from 103.3 to 127.3 nm. FA showed the least bacterial growth reduction %, while the purified fly ash (FA2) showed the highest bacterial growth reduction %, as compared to the control bacterial broth culture without fly ash.