Carbazoles could be easily and rapidly synthesized in a one-pot synthesis using 4-methyl-1-cyclohexanone and p-anisidine in a continuous microwave-heated reaction (1 h, 140 °C). Eustifoline-B, a trace constituent of the roots of Murraya euchrestifolia, was synthesized using the carbazole derivative glycozoline obtained by this method. Next, nine carbazoles isolated from the leaves of M. koenigii and three synthetic carbazoles were evaluated for their antiproliferative activities against human astrocytoma U-251 MG cells [that is, non-cancer stem cells (non-CSCs)] and cancer stem cells (CSCs) isolated by sphere formation. Carbazoles with geranyl or prenyl moieties showed antiproliferative activity against U-251 MG CSCs. In particular, the synthetic compound eustifoline-B showed significant antiproliferative activity against U-251 MG CSCs (IC50 = 2.9 μM). Interestingly, eustifoline-B showed an approximately 10-fold higher antiproliferative activity against U-251 MG CSCs than against U-251 MG non-CSCs (IC50 = 29.4 μM).