The study examined the prevalence of anti-rubella antibodies among pregnant women in South-Western Romania during two periods: 2013–2016 and 2019–2022. A total of 3,268 blood samples were analyzed. The prevalence of acute rubella infection (IgM positive) was low and slightly decreased from 0.54% to 0.50%. The overall prevalence of protective IgG antibodies remained stable (88.28% vs. 88.91%), but fell short of the World Health Organization target of 95%. IgG antibody prevalence increased among the youngest women (under 20 years) and women aged 31–35 years, particularly in rural areas. The results indicate a need to strengthen vaccination campaigns, especially in rural areas and among primary care physicians, to achieve the World Health Organization eradication goal.