Biosecurity is an important part of any successful poultry production system. This study was conducted to assess the biosecurity practices of poultry farmers in commercial poultry farms in Uyo and Ikot-Ekpene LGA of Akwa-Ibom State, Nigeria. Fifty (50) poultry farms were selected for this study and their biosecurity practices were assessed using structured questionnaires administered to the commercial poultry farm owners. Descriptive statistics and inferential statistics such as odd’s ratio at 95% confidence interval and chi-square were used to analyze and present the results. Results of the study revealed that most of the respondents (62%) were males, larger percentage (72%) were married and (36%) were in the age category of 41-50 years. There was no significant association (P>0.05) between age, gender, informal education, and the level of compliance with biosecurity measures. Formal education (secondary and tertiary education) had a significant (P≥0.05) association with the use of biosecurity measures. Non-compliance with biosecurity practices has been related to inadequate training and education of farmers and limited communication between farmers. There is a need for qualified professionals to train poultry farmers, managers, and attendants on proper biosecurity practices.