A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW ("Trp3"), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.