Abstract <p>This study reports the synthesis of 3-(4-cyanophenyl)-5-imino-7-(methylthio)-5<i>H</i>-thiazolo[3,2-<i>a</i>]pyrimidine-6-carbonitrile, achieved by reacting 4-(2-aminothiazol-4-yl) benzonitrile with bis(methylthio)methylene malononitrile in the presence of DMF and anhydrous K₂CO₃. The compound was then subjected to various nucleophiles, including anilines, hetaryl amines, hydrazines, and carbamides, to explore its chemical versatility and potential for structural modification. Spectral and elemental analysis were utilized to confirm the structure of compounds. These newly prepared compounds were screened against both bacterial and fungal strains. Several synthesized compounds exhibited strong antibacterial activity against both Staphylococcus aureus and Escherichia coli, as well as notable antifungal activity against Candida albicans, with some showing better efficacy than the standard drugs streptomycin and fluconazole. Additionally, certain compounds demonstrated antibacterial and antifungal activities comparable to or stronger than the reference drugs. The cytotoxic activity of the synthesized molecules was assessed using the brine shrimp (<i>Artemia salina</i>) lethality bioassay. Molecular docking studies demonstrated strong binding affinities (–8.1 to –9.9 kcal/mol) of the fused thiazole pyrimidine derivatives to bacterial and fungal enzymes, supporting their antimicrobial efficacy.</p>