Synthesis and in vitro antimicrobial activities are reported for a series of 1,3,5-substituted 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indole derivatives. Corresponding pyridazino[4,5-b]indoles were prepared from ethyl 3-formyl-1H-indole-2-carboxylate precursors and the functional group in question was installed with hydrazine and its derivatives. The purity and primary structures of pyridazino[4,5-b]indole were confirmed by IR, H-1 NMR, and C-13 NMR spectroscopy and elemental analyses. All of the indoles were tested for in vitro antimicrobial activity against 8 isolates of bacteria and a fungus including Staphylococcus aureus NRRL B-767, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris NRRL-B123, Salmonella typhimurium NRRL B-4420, Bacillus subtilis NRRL 744, Listeria monocytogenes ATCC 7644, and Candida albicans by using broth microdilution test. All of the isolates showed moderate sensitivity against tested indoles and B. subtilis NRRL 744 was the most sensitive.