Artificial intelligence (AI) has the potential to transform healthcare practices, but the successful adoption of AI depends on the perception and acceptance of healthcare professionals. This study aimed to assess the understanding of AI concepts, recognize the attitude toward AI integration, and identify the barriers to AI adoption among healthcare professionals in a tertiary care hospital. A cross-sectional study was conducted among 200 healthcare professionals, including doctors, nurses, and paramedics, in a tertiary care teaching hospital in Chengalpattu district, India. A semi-structured questionnaire was used to collect data on sociodemographic characteristics, knowledge, attitude, and perceived barriers to AI adoption. The chi-square test was used to analyze the associations between variables. The majorityof the participants, i.e., 54% (n = 108), had moderate knowledge about AI adoption, while 48% (n = 96) had a low attitude toward it.Barriers to the adoption of AI in healthcare practices among healthcare professionals were low among the majority, i.e., 49% (n = 98), of the participants. A statistically significant association was found between knowledge and attitude (X² = 18.052, df = 4, p = 0.001), i.e., healthcare professionals with moderate knowledge levels had low attitudes toward the adoption of AI. A statistically significant association was also found between knowledge and perceived barriers (X² = 31.235, df = 4, p = 0.00), i.e., healthcare professionals with higher knowledge levels perceived lower barriers to the adoption of AI. The study highlights the need for education and training to improve knowledge, foster positive attitudes, and address the perceived barriers to AI adoption among healthcare professionals. Future research should focus on developing targeted interventions to enhance the understanding and acceptance of AI in healthcare practices.