Background Healthcare professionals (HCPs) are the cornerstone of any successful vaccination program. Their knowledge, attitude, and hesitancy are major factors in vaccination uptake. Aim We sought to study the knowledge, attitude, and hesitancy towards the recommended vaccination among healthcare professionals in the Kingdom of Bahrain. Methods A cross-sectional survey was conducted among HCPs at governmental hospitals and primary healthcare centers in the Kingdom of Bahrain. HCPs completed a standardized questionnaire online or in person related to their vaccine knowledge and attitude. Data were weighted for different demographic characteristics. To assess vaccine hesitancy, 15 questions related to vaccine-related attitudes and self-vaccination attitudes were used. Answers to the questions were scored, and the scoring was categorized as low hesitancy, moderate hesitancy, and high hesitancy. Results The study included 552 HCPs. The majority of the participants (75%) believed that vaccines are safe, and 85.5% believed that vaccines protect against infectious diseases. Hepatitis B vaccine and varicella vaccine uptake were less compared to other types of vaccine. None or low vaccine hesitancy was found in 75.2% of the HCPs. Long-term side effects were found to be the highest concern about vaccination in our participants, with 48.4%. 72.5% of participants had not received any training related to vaccination, and 44.1% of those who did had their last training more than 3 years ago. Conclusions Interventions are required to help hesitant HCPs adopt more proactive vaccination practices, and the implementation of policies to check the vaccination status and training is needed urgently.