This study examined the extent to which the impact of a universal professional development (PD) intervention program on children’s early social-emotional learning (SEL) is dependent on early childhood education (ECE) teachers’ stress levels. The program (POMPedaSens) aimed to promote children’s (5–6-year-olds’) SEL by supporting ECE teachers’ PD. Intervention effectiveness was monitored using an 8-month randomized controlled trial design with an intervention group (IG; 26 teachers and 195 children) and a waiting control group (CG; 36 teachers and 198 children) that provided data before and after program implementation. ECE teachers in the IG were trained to implement the intervention program in their early childhood education and care groups. Latent change score analysis revealed that when teachers showed a low level of stress, children’s prosocial behavior increased only in the IG. There were no significant results for the IG in terms of a change in antisocial behavior. The results suggest a promising application of the PD intervention for promoting prosocial behavior in ECE when teachers have low stress. A longer intervention period is likely needed to determine the moderating effect of ECE teachers’ well-being on children’s antisocial behavior change. Unexpectedly, when teachers showed a high level of stress, an increase in prosocial behavior and a decrease in antisocial behavior were found for children in the CG. This could result from demands for accountability and high expectations regarding early prosocial behavior without supporting teachers’ PD and well-being, which can diminish social-emotional functioning in the long run. Decreased antisocial behavior in the CG could stem from an acquired propensity towards compliant behavior driven by either a sense of obedience or fear. High stress in the CG indicates that teachers were trying to do their best at the risk of their own well-being.