Mobile poultry vending and slaughtering of sick poultry have been linked to the spread of highly pathogenic avian influenza (HPAI) H5N1 in Bangladesh. However, limited data exist on associated practices and potential interventions to improve biosecurity among mobile poultry vendors. This mixed-method study was conducted in three phases across four sub-districts in Bangladesh. In phase 1, researchers conducted 416 hours of structured observation, 40 in-depth interviews with poultry vendors, and 40 informal interviews with the customers. Phase 2 involved the development and pilot testing of an intervention package, which included poultry slaughtering cones, hand sanitizers, disinfectants, and a hygiene pamphlet, with 10 vendors. Phase 3 implemented the full intervention with 20 vendors, followed by 94 hours of observation and 17 customer interviews. At baseline, vendors sourced poultry from multiple locations, kept them in small cages on rickshaw vans, and slaughtered them in open spaces, drains, or near water sources. Waste was often discarded in the environment or fed to animals. Vendors demonstrated limited hygiene knowledge and were not observed using personal protective equipment, soap, or disinfectants. Post-intervention, vendors adopted improved practices such as using killing cones, containing waste, disinfecting the slaughtering area, and cleaning hands before eating. Customers also viewed the intervention positively. The study highlights significant risks of AIV transmission through mobile poultry vending but demonstrates that low-cost, targeted interventions can enhance hygiene and biosecurity. Further research is needed to assess long-term sustainability and scalability.