Background: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition for which early diagnosis is critical to ensure timely intervention and improved outcomes. Health care professionals, particularly physicians and nurses, play a key role in the early identification and referral of children with ASD. However, limited data are available regarding ASD-related knowledge among health care workers in Iraq. Objective: This study aimed to determine the level of knowledge regarding ASD among health care professionals working in training hospitals in Al-Diwaniyah, Iraq, and to examine factors associated with knowledge levels. Methods: A descriptive, cross-sectional study was conducted among 447 physicians and nurses working in two training hospitals in Al-Diwaniyah. Data were collected using a Sociodemographic Data Form and the Autism Spectrum Disorder Knowledge Questionnaire. Descriptive statistics and Pearson chi-square tests were used to analyze the data. Results: Overall, 49% of participants demonstrated a moderate level of knowledge regarding ASD, 37.6% had good knowledge, and 13.4% had low knowledge. Knowledge levels were significantly associated with age, gender, marital status, educational level, professional experience, job title, workplace, working pattern, and sources of information (p < 0.05). Younger participants, those with higher education, academic nurses, and health care workers employed at Al-Diwaniyah Teaching Hospital exhibited higher knowledge levels. The internet was the most frequently reported source of ASD-related information, while participation in formal continuing medical education was limited. Conclusion: Health care professionals in Al-Diwaniyah demonstrated a predominantly moderate level of knowledge regarding ASD. Strengthening structured, evidence-based in-service training programs and integrating ASD-focused content into continuing professional education may enhance early recognition and referral, ultimately improving outcome