In the current work, new pyrimidine derivativeswere made from 2-nitrobenzaldehyde, ethyl acetoacetate, and urea and tested for their in vitro antibacterial efficacy against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, two fungus strains, and one Gram-positive strain (Candida albicans and Aspergillus niger). A human breast cancer cell line was tested in vitro using these synthesized compounds (MCF-7). IR and 1H NMR spectrum studies were used to describe the newly synthesized compounds. All synthetic compounds have a significant biological potential against the investigated bacteria, according to the results.