Abstract Professionals’ limited knowledge on mental health and their stigmatizing attitudes toward mental illness can delay the diagnosis of autism. We evaluated the knowledge on Autism Spectrum Disorder (ASD) and stigmatizing attitudes in 93 physicians at Dutch Youth and Family Centers (YFC). These physicians screen for psychiatric symptoms in children. We show that their general ASD knowledge scored 7.1 (SD 1.2), but their specific ASD knowledge was only 5.7 (SD 1.7) (weighted means on 1–10 scale, 1 = least knowledge, 10 = most knowledge). Our physicians had positive attitudes toward mental illness (CAMI scores 2.18 (SD 0.33) to 2.22 (SD 0.40) on a 5-point Likert scale) but they had higher levels of stigmatizing attitudes than other Western healthcare professionals. Their levels were considerably lower than in non-Western professionals. We found no relations between ASD knowledge, stigmatizing attitudes and demographic variables. In conclusion, ASD knowledge and stigmatizing attitudes toward mental illness in Dutch YFC physicians require attention.