The interaction between the HIV-1 transactivator protein Tat and RNA response element (TAR) plays a critical role in HIV-1 transcription. Based on the pharmacophore model of reported inhibitors, a series of novel substituted guanidine indole derivatives were designed, synthesized and evaluated for their in vitro HIV-1 and HIV-2 activity using the IIIB strain and ROD strain, respectively. Preliminary biological evaluation indicated that three compounds exhibited marked inhibitory activity HIV-1 IIIB. Quite unexpectedly, compound a-7 was also endowed with the moderate anti-HIV-2 potency (EC50 = 58.14 μM). In addition, preliminary discussion on the activity results and molecular modeling of these new analogues were presented in this manuscript.