Background: Singapore’s adult vaccination coverage is suboptimal, and this can be attributed to a lack of vaccination recommendations and misconceptions. Studies have explored pharmacists’ vaccination knowledge, attitudes, and practice behaviour overseas but limited information about pharmacists in Singapore is available. This study aims to investigate pharmacists’ vaccination knowledge, attitudes towards providing vaccination services, and their educational needs. Methods: A cross-sectional study was conducted on pharmacists in various practice settings using an anonymous online survey. Results: Pharmacists’ vaccination knowledge (median: 2; IQR: 1–3), confidence in providing vaccination services (median: 6; IQR: 5–8), and frequency of providing vaccination services (median: 2; IQR: 1–3) were only average. However, 74.3% of pharmacists agreed that they play an important role as vaccine advocates. Apparent differences in knowledge level (p = 0.008), confidence level (p = 0.001), attitude (p < 0.001), and the frequency of educating patients (p = 0.001) and recommending vaccinations (p < 0.001) were observed among pharmacists from different practice settings. The main barriers identified were discomfort with giving injections (38.5%) and a lack of skills or knowledge (28.3%) at the point of survey. Conclusions: Pharmacists see the importance of their role as vaccine advocates. However, their vaccination knowledge, confidence in providing vaccination services, and practice behaviours are only average due to inadequate training. Continuous professional education is required to increase pharmacists’ readiness to provide vaccination services.