Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), is a global pandemic causing millions of deaths annually. Highly active antiretroviral therapy (HAART) greatly enhances lifespan but eventually causes debilitating side effects, in part, due to their chronic administration required to suppress HIV-1 replication. If treatment is discontinued, viral suppression is lost and dormant replication-competent monocytic cell reservoirs become reactivated, leading to viral recrudescence and progression to AIDS. Therefore, novel strategies to circumvent obstacles to HIV-1 therapy are critically needed. We evaluated the potentially therapeutic effects of cycloviolacin O2 (CyO2) on cell viability (MTT assay), membrane disruption (SYTOX Green uptake), p24 production [enzyme-linked immunosorbent assays (ELISA)], and proviral integration (PCR amplification) in U1 cells; a monocytic cell model of HIV-1 latency and reactivation. We demonstrate, for the first time, that CyO2 (0.5–5.0 μM) kills productively infected cells. Sub-toxic concentrations (<0.5 μM) of CyO2 disrupted plasma membranes in both latently-infected and productively-infected U1 cells and enhanced the antiviral efficacy of nelfinavir, a HIV-1 protease inhibitor (HPI). Interestingly, CyO2 also decreased virus production by activated U1 cells; however, this effect was not due to suppression of integrated provirus in U1 cells. This suggested that, in addition to the known pore-forming ability of cyclotides, a novel mode of antiviral activity may exist for CyO2. Our data indicate that CyO2 may be a promising candidate for the targeting HIV-1 reservoirs in monocytes, and their inclusion in adjuvant therapy approaches may augment the efficacy of HPIs and ultimately facilitate virus elimination. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 471–479, 2013.