Background and Objectives: The occurrence of human immunodeficiency virus (HIV) infection in children in Romania has been reported since 1989. This retrospective study was aimed at assessing clinical and biological risk factors for mother-to-child transmission (MTCT) of HIV in two HIV-acquired immune deficiency syndrome (AIDS) Regional Centers (RCs), Constanta and Craiova in Romania. Materials and Methods: During the study period (2008–2019), 408 HIV-positive pregnant women, 244 from Constanta RC and 164 from Craiova RC who attended antenatal visits, were included. All HIV-positive pregnant women were under combined antiretroviral therapy (cART) during pregnancy and childbirth, being followedup with their infants up to 18 months after delivery. We investigated the clinical as well as biological risk factorsassociated with increased MTCT of HIV. Results: Comparing different variables of HIV-positive pregnant women from the two HIV-AIDS CRs, we find that there are significant differences between the mean value of hemoglobin, CD4 level, environmental area, marital and amniotic membranes status, and HIV patient stage in the last trimester of pregnancy (p < 0.05), but without any differences in mother’s mean age, education level, type of delivery, breastfeeding, the duration of cART administration, HIV viral load, and survival rate. Conclusions: In 408 HIV-positive pregnant women followed up at two HIV-AIDS RCs in Romania, the most important clinical and biological risk factors associated with increased MTCT of HIV are represented by anemia, CD4 level, and HIV patient stage.