Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against conserved HIV-1 regions. Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication. We show, for the first time, that expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA compared to single shRNA vectors. Thus, their combined expression results in a much stronger inhibition of virus production. Moreover, when we infected cells transduced with a double shRNA viral vector, virus escape was delayed. These results confirm that RNAi has great potential as an antiviral gene therapy approach and support our efforts to develop this strategy for treatment of HIV-1-infected individuals.