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The coupling reaction of diazonium ion of 2-amino-6-nitrobenzothiazole at 0-5 °C with distinctly substituted 2-aminobenzothiazole derivatives produced new 1,2,3,5-tetrazine derivatives. It was found that diazotized 2-amino-6-nitrobenzo[ d]thiazol reacts with the ring nitrogen atom of variously substituted 2-aminobenzothiazole derivatives to yield tetrazine nucleus. The benzene ring of benzothiazole bearing electron donor group and annulated to the tetrazine was further substituted in situ by other (6-nitrobenzo[d]thiazol-2-yl)diazinyl to yield the final product. The structure of the prepared compounds was elucidated using their physical data, elemental analyses, and spectroscopic data. The synthesized compounds were tested for their antimicrobial and antibiofilm activities against Staphylococcus aureus and Escherichia coli bacteria. Two of the synthesis tetrazine derivatives exhibited interesting antibiofilm potential. [ABSTRACT FROM AUTHOR]