Primary healthcare (PHC) workers are central to immunization delivery, yet knowledge and practice gaps continue to undermine the control of vaccine-preventable diseases across sub-Saharan Africa. Nigeria carries the heaviest zero-dose burden on the continent, with over 2.1 million children remaining unvaccinated annually. This study assessed immunization knowledge, attitudes, and practices among PHC workers in rural Kogi local government area (LGA) and urban Lokoja LGA, and identified predictors of immunization practice. A comparative cross-sectional survey enrolled 130 PHC workers through stratified random sampling (Kogi: n = 68; Lokoja: n = 62), using a structured questionnaire and analyzing data with independent-samples t-tests, χ² tests, and linear and logistic regression in SPSS (α = 0.05). Overall, 78.0% of participants demonstrated good knowledge (mean = 7.70 ± 1.55); however, gaps persisted. Only 30.8% correctly identified that freezing inactivates diphtheria-tetanus-pertussis/Hepatitis B/Pentavalent vaccines, and fewer than half (46.9%) recognized that live vaccines are safe for mildly ill children. Knowledge significantly predicted practice (β = 0.192, p = 0.028), though it accounted for only 3.7% of the variance in practice. A multivariable model explained 15.5% of variance, with knowledge (p = 0.028) and rural location (β = 0.198, p = 0.034) as significant independent predictors. Rural workers reported better immunization practices than their urban counterparts (4.20 vs. 3.67; p = 0.034) despite comparable knowledge levels, suggesting that contextual factors, including supervision intensity and workload distribution, shape performance beyond individual knowledge. Prior training was the strongest predictor of good knowledge (odds ratio = 10.52, p = 0.001). These findings show that addressing Nigeria’s immunization gap requires more than training alone; sustained improvements in staffing, supervisory systems, and cold chain infrastructure are equally essential.