The primary objective of this study was to investigate the pattern of antibiotic prescription for prophylaxis purposes among Italian DPs (dental practitioners). A nationwide cross-sectional study was conducted using a multi-stage sampling design. A structured questionnaire was used to collect socio-demographic data and information about antibiotic prophylaxis (AP) prescriptions for selected dental diagnoses and surgical procedures. The presence of an indication and appropriateness of AP were defined according to international guidelines. In total, 563 DPs answered the questionnaire (response rate 52.6%). The proportions of DPs who prescribed AP in the presence of an indication ranged from 39.1% for luxation injury with soft tissue trauma to 73.1% for dental implants, whilst DPs who prescribed AP in healthy patients ranged from 41.9% in luxation injury with soft tissue trauma to 70.3% for bone grafting. The course of AP reported by DPs was not consistent with the guidelines in 70.9% of explored procedures. A high proportion of AP prescriptions before dental procedures were unnecessary. This highlights the urgent need to incorporate recommendations for best practices into national and local protocols as soon as they are established. Specific antibiotic stewardship strategies targeted to DPs should be implemented and assessed for effectiveness in improving prescribing of antibiotics.