Little is known about the prevalence of negative weight-biased attitudes among Dutch healthcare professionals (HCPs) when treating children and adolescents with obesity and whether interdisciplinary differences are present. Accordingly, we asked Dutch HCPs that treat pediatric patients with obesity to complete a validated 22-item self-report questionnaire about their weight-biased attitudes. In total, 555 HCPs participated from seven different disciplines: 41 general practitioners (GPs), 40 pediatricians, 132 youth healthcare physicians, 223 youth healthcare nurses, 40 physiotherapists, 40 dieticians, and 39 mental health professionals. HCPs from all disciplines reported to experience negative weight-biased attitudes among themselves. Pediatricians and GPs scored highest on negative weight-biased attitudes, including frustrations in treating children with obesity, and feeling less confident and prepared to treat children with obesity. Dieticians scored the least negative weight-biased attitudes. Participants from all groups perceived weight bias expressed by their colleagues, toward children with obesity. These findings are comparable to results reported by adult HCPs from other countries. Interdisciplinary differences were found and underscore the need for more research on contributing factors that impact explicit weight bias among pediatric HCPs.