Bacterial infection poses a serious threat to human life due to its rapidly growing resistance to antibacterial drugs, which is a significant public health issue. This study was focused on the design and synthesis of a new series of 25 analogues bearing 5-cyano-6-oxo-4-substituted phenyl-1,6-dihydropyrimidine scaffold hybridized with different substituted benzene-sulfonamides through thioacetamide linker M1-25 via the treatment of the key intermediates 2-mercapto-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitriles 1a-c with different 2-chloro-N-substituted benzene-sulfonamide derivatives 4a-e overnight at room temperature. The spectroscopic data of all the new compounds (IR, 1H-NMR, 13C-NMR, and Mass) was studied in detail to confirm their molecular structures. The antimicrobial activity of the new molecules was studied against various Gram-positive, Gram-negative, and fungal strains. All the tested compounds showed promising broad spectrum antimicrobial efficacy, especially against K. pneumoniae and P. aeruginosa, with ZOI values ranging from 15 to 30 mm. Furthermore, the most promising compounds M6, M19,M 20, and M25 were subjected to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. In addition, the anti-virulence activity of the compounds was also examined using multiple biofilm assays. The new compounds revealed promising suppressing activity of microbial biofilm formation in the examined K. pneumoniae and P. aeruginosa microbial isolates. Additional in silico ADMET studies were conducted to represent their oral bioavailability, drug-likeness characteristics, and human toxicity risks. The obtained data suggest the newly prepared pyrimidine-benzene-sulfonamide derivatives may serve as model compounds amenable for further optimization and development of new antimicrobial and antisepsis candidates.