The spontaneous reporting of suspected adverse events following immunization (AEFI) by healthcare professionals (HCPs) is vital in monitoring post-licensure vaccine safety. The main objective of this study was to assess the knowledge and perceptions of AEFIs among healthcare professionals (HCPs) in Africa, using the situation in Ghana as a case study. The study was of a cross-sectional quantitative design, and was carried out from 1 July 2017 to 31 December 2017 with doctors, pharmacists, and nurses as the study participants. A 28-item paper-based questionnaire, delivered by hand to study participants, was the data collection tool in the study. The study was conducted in 4 hospitals after ethical approval was granted. The desired sample size was 686; however, 453 consented to partake in the study. Data were analyzed using SPSS (software version 22, IBM, Armonk, NY, USA), and chi-square and binary logistic regression tests were used for tests of association between HCPs’ characteristics and their knowledge and perceptions. Detailed knowledge of AEFIs was ascertained with a set of 9 questions, with 8 or 9 correctly answered questions signifying high knowledge, 5 to 7 correctly answered questions signifying moderate knowledge, and below 5 correctly answered questions signifying low knowledge. A set of 10 questions also ascertained HCPs’ positive and negative perceptions of AEFI. Results revealed that knowledge of AEFIs was high in 49 (10.8%) participants, moderate in 213 (47.0%) participants, and low in 191 (42.2%) participants. There was no statistically significant correlation between AEFI knowledge and professions. The highest negative perception was the lack of desire to learn more about how to diagnose, report, investigate, and manage AEFI, whereas the lowest was the lack of belief that surveillance improves public trust in immunization programs. There was a general awareness of AEFIs among HCPs in this study. However, negative perceptions and the lack of highly knowledgeable HCPs regarding AEFIs were possible setbacks to AEFI diagnosis, management, prevention, and reporting. More training and sensitization of HCPs on AEFIs and vaccine safety will be beneficial in improving the situation. Future research should focus on assessing the training materials and methodology used in informing HCPs about AEFIs and vaccine safety.