Avian influenza (AI) is a severe respiratory disease affecting wild and domestic birds globally. There is currently no approved vaccine for AI control in Nigeria. Therefore, biosecurity measures remain the primary preventive strategy. However, there is limited information on the AI carrier status of captive wild birds and the implementation of biosecurity programs (BPs) in zoological gardens across the country. This study aimed to screen captive wild birds for AI and evaluate the BPs in selected zoological gardens in southwestern Nigeria. Using a cross-sectional approach, cloacal swabs and freshly deposited faecal samples (n?=?149) were collected from captive wild birds in seven zoological gardens in southwestern Nigeria following an AI outbreak in 2022. The samples were screened for AI viruses using RT-qPCR, and BPs in 13 consenting zoological gardens were assessed using a structured questionnaire. The responses were scored, and the BPs were categorised as ‘Poor’, ‘Fair’, or ‘Good’. Descriptive and inferential statistical methods were used to analyse the data. All samples tested negative for AI viruses. Documented institutional biosecurity and disease outbreak contingency plans were available in 84.6% of the zoos. Most of the zoos demonstrated good BPs across key categories, including vehicle and animal movement control (100%), food and water supply management (92.3%), enclosure hygiene, ground maintenance and waste disposal (84.6%), pest control (76.9%), and quarantine and sick animal management (69.2%). These findings suggest that the captive wild birds in zoological gardens in southwestern Nigeria are unlikely to serve as AI reservoirs, and most of the zoos have robust BPs that contribute to preventing AI and other avian diseases.