Introduction: In Guinea, vaccination coverage remains low despite various initiatives. This study assessed the prevalence of parental hesitancy toward routine childhood immunization in the Matoto health district and identified factors associated with this hesitancy, a key barrier to improving vaccination uptake. Methods: An analytical cross-sectional survey was conducted in June 2025 in the Matoto health district, targeting parents of children under five years of age. The SAGE group’s WHO questionnaire (10 items) was used to determine the proportion of parents hesitant toward vaccination. Factors associated with hesitancy were assessed using multivariate logistic regression. Results: Among 429 parents surveyed, 21.2% expressed vaccine hesitancy. Factors significantly associated with higher hesitancy included lack of formal education (AOR = 2.21; 95% CI: 1.51 - 3.61), having a child with an adverse event following immunization (AOR = 2.67; 95% CI: 1.34 - 4.26), residing over 5 km from a vaccination center (AOR = 1.87; 95% CI: 1.20 - 3.22), and experiencing poor reception at health facilities (AOR = 1.54; 95% CI: 1.10 - 2.28). Conversely, lower hesitancy was linked to attending at least four antenatal visits (AOR = 0.29; 95% CI: 0.11 - 0.42), receiving follow-up from qualified health personnel during pregnancy (AOR = 0.32; 95% CI: 0.21 - 0.53), and delivering in a health facility (AOR = 0.49; 95% CI: 0.34 - 0.67). Conclusion: The findings highlight a substantial level of parental vaccine hesitancy in the Matoto health district. Developing and implementing context-specific and targeted strategies is essential to reduce hesitancy and improve routine immunization coverage. Future research should examine the impact of digital media on vaccine confidence in this population.
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