Background: Toxoplasma gondii is a globally prevalent parasite with significant health implications, particularly for pregnant women and immunocompromised individuals. Infections during pregnancy can result in serious outcomes such as miscarriage, stillbirth, or congenital disabilities. This study aimed to assess the prevalence of T. gondii infection among pregnant women and small ruminants in Al-Anbar province, Iraq, using serological and molecular diagnostic techniques. Methods: Blood samples were collected from 165 pregnant women and 100 small ruminants (sheep and goats) between January and May 2021. Serological screening was conducted using the Humatex-Toxo rapid test to detect antibodies against T. gondii. DNA was extracted from the blood samples, and nested polymerase chain reaction (PCR) targeting the T. gondii B1 gene was used for molecular detection. The infection rates were analyzed statistically using ANOVA and Chi-square tests, with significance set at p<0.05. Results: The serological test identified T. gondii antibodies in 32.7% of pregnant women, while the PCR detected a higher infection rate of 40%. Among the small ruminants, PCR revealed an overall infection rate of 10%, with sheep showing a higher rate (16%) compared to goats (4%). The results indicate that PCR is more sensitive than serological testing, particularly for identifying active infections. Additionally, the higher infection rate in young women may be linked to lifestyle, environmental exposure, and dietary habits, while seasonal factors likely play a role in infection distribution. Conclusion: Nested PCR is a more effective method for detecting T. gondii in pregnant women, offering a precise tool for identifying infections and potential causes of abortion. The lower infection rates in small ruminants suggest a reduced zoonotic risk in the study area, although continued monitoring is essential. Negative PCR results may be due to inhibitors in the DNA extraction process or the parasite’s localization in tissues rather than blood. The findings highlight the importance of molecular diagnostics in public health strategies aimed at reducing T. gondii infections in high-risk populations.