This cross-sectional study aimed at evaluating the knowledge and attitudes concerning influenza vaccination in Southern Italy, and investigating the potential determinants of vaccine uptake. The sample consisted of 700 adults (mean age 58.7y) with chronic diseases attending four public specialty clinics in Italy. Overall, 64.7% of the participants were aware that influenza can be prevented with vaccines and that patients with chronic diseases are at higher risk of developing severe complications. Less than half of the sample (42.1%) received influenza vaccine in the last season, and 46.9% declared the will to receive influenza vaccination in the next season. The level of awareness was significantly lower among the elderly (> = 65y) and those with a higher self-reported health. A significantly higher likelihood of vaccination was observed among the elderly, the subjects with a higher knowledge about vaccine utility and safety, the participants with chronic respiratory diseases, and those who had taken more drugs. Future education programs and communication strategies are strongly needed in adults with chronic diseases to improve influenza vaccination knowledge and uptake.