Background: In vaccine delivery, a health worker (HW), particularly primary care physicians’ knowledge and behavior in handling AEFI on COVID-19 vaccine are important. The role of health workers and non-health workers in building public trust in vaccination programs is essential in the community. Research Objective: Comparing the knowledge and behavior in handling the AEFI vaccine COVID-19 between health workers and non-health workers at a hospital in Kupang. Settings and Design : This is a cross-sectional observational analytic study conducted on health workers and non-health workers at a hospital in Kupang. Method and Material: There are 118 respondents who met the inclusion criteria that were chosen through the consecutive sampling using validated and reliable questionnaires to assess knowledge, and behavior on handling vaccine AEFI. Statistical analysis used: The research was analyzed univariately using a frequency distribution table and bivariate analysis using the chi-square test. Results: In terms of knowledge, 60.5% of health workers had sufficient knowledge, while 56.3% of non-health workers had enough knowledge. In terms of behavior, 74.4% of health workers and 62.5% non health workers behaved well. The results of the bivariate test using the Chi-Square test for the analysis of differences in knowledge obtained p = 0.432 (p>0.05), while for the study of behavioral differences, the results were p = 0.297 (p>0.05). Conclusion: It is important to monitor the level of understanding of AEFI among health and non-health workers, as they play a significant role in establishing public trust.