BACKGROUND: COVID-19 vaccines are highly effective in preventing severe disease and death but are underused. Interventions to address COVID-19 vaccine hesitancy are paramount to reducing the burden of COVID-19. OBJECTIVE: We aimed to evaluate the preliminary efficacy, usability, and acceptability of a chatbot for promoting COVID-19 vaccination and examine the factors associated with COVID-19 vaccine hesitancy. METHODS: In November 2021, we conducted a pre-post pilot study to evaluate "Vac Chat, Fact Check," a web-based chatbot for promoting COVID-19 vaccination. We conducted a web-based survey (N=290) on COVID-19 vaccination at a university in Hong Kong. A subset of 46 participants who were either unvaccinated (n=22) or were vaccinated but hesitant to receive boosters (n=24) were selected and given access to the chatbot for a 7-day trial period. The chatbot provided information about COVID-19 vaccination (eg, efficacy and common side effects), debunked common myths about the vaccine, and included a decision aid for selecting vaccine platforms (inactivated and mRNA vaccines). The main efficacy outcome was changes in the COVID-19 Vaccine Hesitancy Scale (VHS) score (range 9-45) from preintervention (web-based survey) to postintervention (immediately posttrial). Other efficacy outcomes included changes in intention to vaccinate or receive boosters and willingness to encourage others to vaccinate on a scale from 1 (not at all) to 5 (very). Usability was assessed by the System Usability Scale (range 0-100). Linear regression was used to examine the factors associated with COVID-19 VHS scores in all survey respondents. RESULTS: The mean (SD) age of all survey respondents was 21.4 (6.3) years, and 61% (177/290) of respondents were female. Higher eHealth literacy (B=-0.26; P<.001) and perceived danger of COVID-19 (B=-0.17; P=.009) were associated with lower COVID-19 vaccine hesitancy, adjusting for age, sex, chronic disease status, previous flu vaccination, and perceived susceptibility to COVID-19. The main efficacy outcome of COVID-19 VHS score significantly decreased from 28.6 (preintervention) to 24.5 (postintervention), with a mean difference of -4.2 (P<.001) and an effect size (Cohen d) of 0.94. The intention to vaccinate increased from 3.0 to 3.9 (P<.001) in unvaccinated participants, whereas the intention to receive boosters increased from 1.9 to 2.8 (P<.001) in booster-hesitant participants. Willingness to encourage others to vaccinate increased from 2.7 to 3.0 (P=.04). At postintervention, the median (IQR) System Usability Scale score was 72.5 (65-77.5), whereas the median (IQR) recommendation score was 7 (6-8) on a scale from 0 to 10. In a post hoc 4-month follow-up, 82% (18/22) of initially unvaccinated participants reported having received the COVID-19 vaccine, whereas 29% (7/24) of booster-hesitant participants received boosters. CONCLUSIONS: This pilot study provided initial evidence to support the efficacy, usability, and acceptability of a chatbot for promoting COVID-19 vaccination in young adults who were unvaccinated or booster-hesitant.