The attitude of professionals towards victims’ disclosures of child sexual abuse has a decisive influence on the well-being of the victims. However, both vignette studies as well as victim surveys suggest that professionals’ perception on sexual abuse is influenced, among other factors, by the perpetrators’ gender. The present analysis investigates whether professionals’ perception of sexual abuse is influenced by the perpetrators’ gender, the professionals’ gender as well as their experience and field of profession. Additionally, the effect of an e-learning course was examined. As part of an e-learning course on child sexual abuse, N = 1925 German professionals (social work, educational, medical-therapeutic, legal professionals) were presented with pre- and post-editing vignettes differing in the perpetrators’ gender and the ambiguity of the situation. The professionals had to rate the situations in terms of sexual abusiveness. The findings revealed that the same situation would be more likely classified as abusive when the perpetrator was male, particularly in ambiguous situations. Moreover, work experience, personal experiences with sexual abuse, and awareness of sexual abuse had significant positive relationships with the professionals’ rating of the abusiveness of a situation. An effect of training was identified, with situations being more likely rated as abusive after the course, specifically in cases with female perpetrators. Victims of female-perpetrated sexual abuse are still often disbelieved, and their experiences trivialized because professionals perceive violence by women differently due to gender stereotypes and a lack of knowledge on female-perpetrated violence. This study highlights the important effect of training. Both science and professional practice need to increase knowledge about female-perpetrated child sexual abuse, as it remains a taboo topic.