6'-(4-Chlorophenyl)-spiro[cyclohexane-1,2'-thieno[3,2-d][1,3] oxazin]-4'(1'H)-one (1) was synthesized and used as a starting material for the synthesis of a novel series of spiro compounds having biologically active sulfonamide 2a-e and 3'-(4-acetylphenyl)-6'- (4-chlorophenyl)-1'H-spiro[cyclohexane-1,2'-thieno[3,2-d] pyrimidine-4'(3'H)-one (3). Compound 2a was used as a key intermediate for the synthesis of sulfonyl carbothioamide derivatives 4a-c. Also, compound 3 was used as an intermediate for the synthesis of 3'H-spiro[cyclohexane-1,2'-thieno[3,2-d]pyrimidin]-3'-yl] phenyl}-2-imino-4-(substituted phenyl and/or thienyl)-1,2-dihydropyridine- 3-carbonitrile derivatives 5a-e, 3'H-spiro[cyclohexane- 1,2'- thieno[3,2-d]pyrimidin]-3'-yl]phenyl}-2-oxo-4-(substituted phenyl and/or thienyl)-1,2-dihydropyridine-3-carbonitrile derivatives 6a-e, and 4-[(2Z)-3-substituted-arylprop-2-enoyl] phenyl-1'H-spiro[cyclohexane-1,2'-thieno[3,2-d]pyrimidine derivatives 7a-e. Cyclocondensation of 7a-e with hydrazine hydrate produced 6'-(4-chlorophenyl)-3'-[4-(5-substituted aryl-4,5-dihydro- 1H-pyrazol-3-yl)phenyl]-1'H-spiro[cyclohexane-1,2'-thieno- [3,2-d]pyrimidin]-4'(3'H)-ones 8a-e but with hydroxylamine hydrochloride afforded the corresponding isoxazoline derivatives 9a-e. Also, cyclocondensation by thiourea afforded 2-thioxo-1,2- dihydropyrimidin-4-yl)-phenyl-spiro-{cyclohexanethieno[3,2-d] pyrimidin}-4-one derivatives 10a-e. The new compounds were investigated for antimicrobial activity. Compounds 2c, 8b,c, 9b and 10b were the most potent ones against both Gram-negative and Gram-positive bacteria. Compound 8c exhibited higher antifungal activity towards the examined fungi with MIC of 1-2 μmol mL-1 compared to ketoconazole (MIC 2-3 μmol mL-1 ).