Cerium oxide (CeO2) nanoparticles (NPs) have a wide range of biological and biomedical applications. This work describes a new methodology for producing ultrafine, highly uniform NPs with controlled sizes using the chemical microwave assisted route. The size of CeO2-NPs decreased from 10 to 5 nm by increasing the molar ratio of cerium nitrate Ce(NO3)3.(6H2O) to that of hexamethylenetetramine (C6H12N) from 1:20 to 20:20. Detailed information about their structural characterization was obtained from the XRD, UV-visible, photoluminescence, Raman spectroscopy, SEM, TEM and AFM. These CeO2-NPs were tested as antimicrobial agent against Gram-negative (Escherichia.coli), Gram-positive (Bacillus.subtilis) bacteria and yeast (Saccharomyces cerevisiae). The obtained results showed significant inhibition of these strain even at low concentration of CeO2-NPs. The CeO2-NPs with the molar ratio 5:20 had the most effective inhibition against E.coli (~70%) at a concentration of 20 µL. The CeO2-NPs with the ratio 12:20 were found to be the most effective against B.subtilis (inhibition ~68%). On the other hand, CeO2-NPs synthesized with the 20:20 molar ratio caused the highest inhibition for S. cerevisiae (~60%). It is observed that at higher NPs concentration (i.e., >20 µL) the inhibition of these strains decreased. The antimicrobial activity may be attributed to the penetrating power of CeO2-NPs size beside the generated oxygen species radicals that caused inhibition of bacterial growth.