Lack of knowledge is the major reason for non-compliance with correct healthcare-associated infections (HAI) prevention procedures. The aim of this study was to evaluate knowledge of the Dental School (DSS) and Dental Hygiene (DHS) students with regard to the prevention of HAI, as basic knowledge for improving and harmonizing the educational content in the different Italian Universities. A cross-sectional study was carried out using an anonymous questionnaire that was completed by DSS (I, II, III, IV, and V year) in seven Universities and DHS (I, II, and III year) in three Universities. The questions dealt with three specific areas: healthcare-associated infections, standard precautions and hand hygiene. Factors associated with an unacceptable level of knowledge (score <17.5) were analyzed using a logistic regression model. A p value <0.05 was considered to be significant. Five hundred and four questionnaires were collected: 81.5% for DSS and 18.5% for DHS. Mean overall score (±DS) achieved by the total number of students was 18.2±2.93 on an overall perfect score of 25; 18.2±3.04 for DSS and 17.8±2.31 for DHS. Stratifying by area, the average score 2.7±1.07 (53%) for HAI, 10.3±1.61 (85.9%) for standard precautions, and 5.2±1.44 (64.8%) for hand hygiene was observed. A significantly different level of knowledge (p<0.001) between DSS and DHS was observed only for HAI (2.8±1.07 for DSS vs 2.1±0.96 for DHS). Significant differences among the academic years were found only for DSS concerning HAI and standard precautions. The logistic regression model showed that an age <23 years was a risk factor for lack of knowledge on HAI, but a protective factor for lack of knowledge about standard precautions and hand hygiene; attending DH degree course was associated with lack of knowledge on HAI. Although the overall score obtained both by DSS and DHS indicated an acceptable level of knowledge, lack of knowledge was highlighted, in particular, for hand hygiene. Therefore, it is necessary to implement and validate effective teaching models in undergraduate courses in order to provide the scientific basis and the theoretical and practical preparation for the prevention and control of HAI.