Stroke is a global leading cause of death and disability. Knowledge of related guidelines is crucial for emergency department (ED) staff, influencing early diagnosis and timely treatment. We investigated Greek Cypriot ED healthcare professionals' (nurses and physicians) knowledge in recognizing and managing stroke. A descriptive cross-sectional study spanned November 2019 to April 2020, encompassing four private and seven public EDs in the Republic of Cyprus. The data were collected through a self-reported questionnaire developed by the research team, consisting of 37 questions. Eight questions focused on sociodemographic and employment characteristics, twenty-eight assessed knowledge in stroke recognition and management (each item was equally weighted without deliberate prioritization), and one question addressed self-assessment of knowledge in stroke care. A total of 255 nurses (response rate (RR): 74.1%) and 26 physicians (RR: 47.3%) completed the questionnaire. The average correct response rate was 12.9 out of 28 statements (SD: 4.2), with nurses and physicians scoring 12.6 (SD: 4.1) and 15.7 (SD: 4), respectively. Work experience significantly influenced stroke knowledge, with all groups demonstrating superiority over those with less than one year of experience. Participants with previous training scored an average of 1.45 additional correct answers while educational attainment did not significantly influence stroke knowledge. Investigating stroke knowledge among emergency department nurses and physicians in the Republic of Cyprus revealed significant deficits. This study stresses targeted interventions, including education, yearly examinations, workshops with hands-on training, and repeated training, to address these gaps and enhance the overall stroke care capabilities of the healthcare professionals.