Poultry farming is an important socio-economic sector in Burkina Faso. However, disease outbreaks can substantially impact livestock production, so appropriate preventive measures should be implemented. The present study aimed to assess the implementation of biosecurity measures in poultry farms in the Haut-Bassins Region, Burkina Faso. The present cross-sectional study included 79 poultry farms and used a validated questionnaire. Questions were asked of each poultry farmer through interviews, and farms were observed to verify the implementation of certain biosecurity measures, including the presence and quality of foot baths, the presence of stagnant water, the orientation of the buildings, and the presence of gates. The current results indicated that the majority of farms were managed by men (86.1%), and 45.6% of farmers had more than 10 years’ experience. Regarding farm capacity, 68.4% could house up to 10,000 poultry, and 64.6% of the reported farms had facilities that could accommodate more than 1,000 poultry. Over 59.5% of farms were located more than 300 meters away from the main road; however, 64.6% were located less than 500 meters from the nearest poultry farm. More than 97.5% of farms were enclosed by fences, and 87.3% of these fences were made from waterproof materials. On farms, 60.8% had functional footbaths, 11.4% produced poultry feed, and 92.4% were not protected against wild birds entering their airspaces. Most farms (97.5%) regularly cleaned and disinfected materials and equipment, but only 38% had suitable waste disposal practices, and 22.8% kept water waste outside the farm. The present study reported that 56.9% of farms implemented a satisfactory level of biosecurity. Farmers who attended poultry farming training were more likely to apply biosecurity practices on their farms. The present study delineated the application of biosecurity measures on farms and emphasized the importance of raising awareness about biosecurity among poultry farmers in the Haut-Bassins Region of Burkina Faso.