A series of 1,3-indanedione derivatives were synthesized and evaluated as antimicrobial agents using 2-amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (2) as a starting material. Compound 2 reacted with various types of reagents under different conditions to yield thienopyrazole 3, thienopyrimidines (6, 8, 9), thienopyridines (10, 11), and thiazolylidines (15, 17, 19, 21, 23). These compounds were screened for their antibacterial activity against gram-positive bacteria (Bacillus subtilis and Bacillus thuringiensis) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and antifungal activity against Fusarium oxysporum and Botrytis fabae strains. 2-((3,4-Diphenylthiazol-2(3H)-ylidene) amino)-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (15) and 8-oxo-2-((4-oxo-3-phenylthiazolidin-2-ylidene) amino)-8H-indeno[2,1-b]thiophene-3-carbonitrile (17) were found to exhibit equal activity compared with chloramphenicol against B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than that of chloramphenicol against B. thuringiensis.