Background: Gut microbiota can be associated with COVID-19 vaccine immunogenicity. We investigated whether recent antibiotic use influences BNT162b2 vaccine immunogenicity. Methods: BNT162b2 recipients from three centers were prospectively recruited. Outcomes of interest were seroconversion of neutralising antibody (NAb) at day 21, 56 and 180 after first dose. We calculated the adjusted odds ratio (aOR) of seroconversion with antibiotic usage (defined as ever use of any antibiotics within six months before first dose of vaccine) by adjusting for covariates including age, sex, smoking, alcohol, and comorbidities. Results: Of 316 BNT162b2 recipients (100 [31.6%] male; median age: 50.1 [IQR: 40.0–57.0] years) recruited, 29 (9.2%) were antibiotic users. There was a trend of lower seroconversion rates in antibiotic users than non-users at day 21 (82.8% vs. 91.3%; p = 0.14) and day 56 (96.6% vs. 99.3%; p = 0.15), but not at day 180 (93.3% vs. 94.1%). A multivariate analysis showed that recent antibiotic usage was associated with a lower seroconversion rate at day 21 (aOR 0.26;95% CI: 0.08–0.96). Other factors associated with a lower seroconversion rate after first dose of the BNT162b2 vaccine included age ≥ 60 years (aOR: 0.34;95% CI: 0.13–0.95) and male sex (aOR: 0.14, 95% CI: 0.05–0.34). There were no significant factors associated with seroconversion after two doses of BNT16b2, including antibiotic use (aOR: 0.03;95% CI: 0.001–1.15). Conclusions: Recent antibiotic use may be associated with a lower seroconversion rate at day 21 (but not day 56 or 180) among BNT162b2 recipients. Further long-term follow-up data with a larger sample size is needed to reach a definite conclusion on how antibiotics influence immunogenicity and the durability of the vaccine response.