Background. Smallholder farmers have a significant role in contributing to improving the establishment and effectiveness of biological control agents through their on-farm management practices. Biological control offers a long-term sustainable solution but requires farmer engagement and support to ensure effectiveness through a coordinated area-wide management approach. The stem-boring weevil, Listronotus setosipennis, has been approved for release as a classical biological control agent for Parthenium hysterophorus L. (Asteraceae) (“parthenium”), a major weed in Pakistan. The objective of this study was to investigate men and women smallholder farmers’ knowledge, attitudes and practices towards parthenium and biological control. Methods. Household surveys using a structured questionnaire were conducted with 562 farmers (62% men and 38% women) in Khyber-Pakhtunkhwa (KP), Punjab and Azad Jammu and Kashmir (AJK). Stratified random sampling was used to select participants from distinct districts within each of the three locations. Seven focus group discussions were conducted with separate men’s and women’s groups in each district and key informant interviews were conducted with 17 extension agents and 31 agro-dealers. Results. In all survey areas, women and men farmers reported parthenium present on their farming land, abundant on common land, and rapidly spreading. Women reported higher levels of land covered by parthenium, likely due to their frequent presence within the field during hand weeding. Women farmers and farmers in KP, more frequently reported negative health effects due to contact with parthenium. The two key management methods used for parthenium were hand weeding and chemical herbicides, the former used more frequently by women. Extension agents have an important role in advising farmers and promoting the use of alternatives such as biological control, but women do not engage in many extension activities. Conclusions. Biological control of parthenium using L. setisopennis offers an opportunity to engage with farmers and strengthen their knowledge of biological control while demonstrating its use at a landscape level. Targeted awareness campaigns using face-to-face and other verbal methods of communication are recommended. Increasing women’s access to information would improve their knowledge of parthenium, effective management approaches, and ways to protect their own and their family members health.