Background: Neonatal mortality remains high in low- and middle-income countries. Mothers’ knowledge of Essential Newborn Care (ENC) and neonatal danger signs is crucial for promoting appropriate neonatal care, facilitating early recognition of illness, and reducing neonatal mortality. Structured health education may improve maternal knowledge; however, evidence of its effectiveness in routine hospital settings remains limited. Objective: To evaluate the effectiveness of health education intervention on postnatal mothers’ knowledge of ENC and neonatal danger signs. Methods: A quasi-experimental pre–post study using independent cross-sectional samples was conducted among postnatal mothers in four public hospitals in Dar es Salaam, Tanzania. The post- intervention survey (2021) included 825 mothers and was compared with a pre-intervention sample of 825 mothers from 2019. A structured maternal health education package was implemented between the two time points. Data were collected using structured questionnaires assessing maternal knowledge of ENC and neonatal danger signs. Descriptive statistics and logistic regression analyses were performed, adjusting for sociodemographic and care-related confounders. Statistical significance was set at P<.05. Results: Post-intervention mothers had higher levels of education (34.7% vs 28.7%. P=.001), employment (10.2% vs 5.2%; P=.001), and caesarean delivery (30.9% vs 21.9%; P=.025), but lower uptake of ≥4 antenatal care visits (81.0% vs 85.4%; P=.003). A higher proportion of post- intervention mothers had heard of neonatal danger signs (92.4% vs 85.8%; P<.001) and correctly identified at least 50% of them (41.6% vs 36.7%; P=.044). They were more than twice as likely to correctly identify danger signs (adjusted OR, 2.38; 95% CI, 1.63–3.48). Maternal knowledge was also positively associated with ≥4 antenatal care visits, receipt of postnatal health education, and delivery at lower volume hospitals. Conclusion: The structured health education intervention was associated with higher maternal knowledge of neonatal danger signs and ENC, although gaps remained in breastfeeding and some infection prevention practices. Knowledge was greater among mothers with four or more antenatal care visits, those delivering at lower-volume hospitals, and those exposed to postnatal education. Future longitudinal studies are needed to assess knowledge retention and how it translates into actual newborn care practices.