Objective: To assess healthcare workers’ knowledge and attitudes about human papillomavirus (HPV) infection, related diseases, and prevention. Methods: A cross-sectional multicenter survey about HPV and its prevention, targeted to healthcare workers involved in HPV vaccine counseling, was performed from May 2017 to December 2018. Results: The overall median knowledge and attitude scores were 69.2% (25–75, p = 61.5–84.6) and 5 (25–75, p = 4–5), respectively. Both knowledge and attitudes statistically significantly differ between physicians and healthcare professions. The median propensity score before and after the educational intervention was stable and high, at 10 (25–75, p = 9–10). The predictors of statistically significantly high knowledge scores are to be a physician, general practitioner, or pediatrician, attending courses/congresses, and consulting technical product characteristics and scientific literature to obtain information about the HPV vaccine. Being a physician and consulting scientific literature to obtain information about the HPV vaccine were found also as predictors of statistically significantly different attitude scores among study participants. Conclusions: Although healthcare workers showed overall positive attitudes towards the relevance of HPV burden and prevention tools, demonstrated knowledge was largely suboptimal, particularly that shown by healthcare professions. Obtained results allow highlighting knowledge gaps, and thus improving counselling to HPV vaccine targets.