The study aimed to investigate the assessment of farm profiles, knowledge gain, and adoption behaviours of biosecurity practices following a training intervention in commercial poultry farms in Tamil Nadu. A total of 89 farmers from commercial desi (32), layer (30), and broiler (27) farms participated in the training program. The biosecurity assessment used an evaluation framework with a two-day training program. Participants completed pre- and post-training surveys to measure knowledge gained during the training. Additionally, we conducted a follow-up evaluation of adoption behaviours after 90 days of training intervention. Questionnaire data were analysed using paired sample t-test, chi-square, and regression analysis. Results revealed that 89% of the trainees were male, 90% were in the age group between 21 and 60 years, and 88% had a secondary education level or higher. Further, 56.2% of farmers had 5-20 years of experience in poultry farming, and 46.1% of farmers revealed that the significant source of income is from poultry farming and agricultural practices. A pre- and post-survey data comparison showed that all the farmers had significant knowledge gain (p < 0.01) in all the categories of structural and operational biosecurity practices immediately after the training. The farmer's educational qualification significantly influences the knowledge gain except for dead bird disposal (p < 0.05). Commercial desi and layer farmers have more pre-existing knowledge compared to broiler farmers. The broiler farmers showed the highest knowledge gain compared to layer and commercial desi farmers. Still, there was no significant difference between knowledge gain among different types of poultry farmers (p > 0.05). The adoption behaviour measured after 90 days significantly increased in all categories (p < 0.05) except for rodent and pest control. This comprehensive study provided valuable insights regarding farmers' existing knowledge and the impact of training on some behavioural changes to improve biosecurity. The study concluded that a tailored training program is essential to educate small-scale producers about biosecurity measures to prevent poultry food-borne diseases.