West Nile virus (WNV) has progressively endemized in large areas of continental Europe, and particularly in Northern Italy, in the Po River Valley. During summer season 2022, Italy experienced an unprecedented surge in incidence cases of WNV infections, including its main complications (West Nile fever (WNF) and West Nile neuroinvasive disease (WNND)). As knowledge, attitudes, and practices (KAP) of medical professionals may be instrumental in guaranteeing a prompt diagnosis and an accurate management of incident cases, we performed a cross-sectional study specifically on a sample of Italian medical professionals (1 August 2022–10 September 2022; around 8800 potential recipients). From a total of 332 questionnaires (response rate of 3.8%), 254 participating medical professionals were eventually included in the analyses. Knowledge status of participants was unsatisfying, as most of them exhibited knowledge gaps on the actual epidemiology of WNV, with similar uncertainties on the clinical features of WNF and WNND. Moreover, most of participants substantially overlooked WNV as a human pathogen when compared to SARS-CoV-2, TB, and even HIV. Interestingly, only 65.4% of respondents were either favorable or highly favorable towards a hypothetical WNV vaccine. Overall, acknowledging a higher risk perception on WNV was associated with individual factors such as reporting a seniority ≥ 10 years (adjusted odds ratio [aOR] 2.39, 95% Confidence interval [95%CI] 1.34 to 4.28), reporting a better knowledge score (aOR 2.92, 95%CI 1.60 to 5.30), having previously managed cases of WNV infections (aOR 3.65, 95%CI 1.14 to 14.20), being favorable towards a hypothetic vaccine (aOR 2.16, 95%CI 1.15 to 4.04), and perceiving WNV infections as potentially affecting daily activities (aOR 2.57, 95%CI 1.22 to 5.42). In summary, substantial knowledge gaps and the erratic risk perception collectively enlighten the importance and the urgency for appropriate information campaigns among medical professionals, and particularly among frontline personnel.