Antiretroviral therapy has made HIV a chronic infectious disease. In Sweden, assisted reproductive technologies using sperm washing were implemented in 2004 for treatment of all HIV-positive males in serodiscordant couples at Karolinska Hospital in Stockholm. This study aimed to assess efficacy and safety of this approach and to investigate the effect of antiretroviral regimens on sperm quality of men treated for HIV. The study included all HIV-positive men ( = 53) adherent to antiretroviral treatment, including nucleoside/nucleotide analog reverse-transcriptase inhibitors (NRTIs) combined with non-nucleoside reverse-transcriptase inhibitors (NNRTIs) in 22 cases, or with other medication in 31 cases wishing fertility treatment. Sperm was washed using gradient centrifugation with a double tube system, frozen and thawed. Thereafter, single spermatozoa were injected in oocytes by intracytoplasmic sperm injection (ICSI). Treatment outcomes were compared to those of hepatitis B virus (HBV)-seropositive men in serodiscordant couples ( = 56), undergoing similar sperm washing protocols and ICSI at the center. In total, 82 fresh and 39 frozen/thawed embryo transfer (FET) cycles were performed in discordant HIV couples and 92 fresh and 36 FET cycles in HBV. Fertilization rates (69.2% vs. 64.6%,  = 0.310), clinical pregnancy rates (39.2% vs. 32.6%,  = 0.378), and live birth rates (35.8% vs. 31.5%,  = 0.565) were similar between the groups and the cumulative results yielded similar outcomes with a live birth rate of 52.8% and 51.8% ( = 0.913), respectively. No post-wash sperm sample was found positive for HIV viral particles. No partner seroconversion or offspring infection was reported. NNRTI-treated men had significantly lower motile sperm concentration than non-NNRTI-treated patients (54.48% vs. 36.41%,  < 0001). This study showed that fertilization/ICSI treatment with sperm washing was safe in individuals compliant to antiretroviral treatment and the reproductive outcome was similar to that of HBV-positive men. The observed moderate negative impact on sperm motility of NNRTI regimens may be overcome by using ICSI.