Background: Both healthcare-associated infections (HAIs) and antimicrobial resistance are associated with an increased length of stay and hospital costs, while they have also been linked to high morbidity and mortality rates. In 2016 and 2017, the latest point prevalence survey (PPS) of HAIs and antimicrobial use in European acute care hospitals highlighted an HAI prevalence of 6.5%, while Greece had a higher HAI prevalence of 10%. The aim of this PPS was to record the prevalence of HAIs and antimicrobial use in all eight public acute care hospitals in Crete, Greece during the COVID-19 pandemic in order to highlight the types of infections and antimicrobial practices that need to be prioritized for infection control initiatives. Methods: The PPS was conducted between 30 March and 15 April 2022, according to the ECDC standardized relevant protocol (version 5.3). Statistics were extracted using the ECDC Helics.Win.Net application (software version 4.1.0). Results: A total of 1188 patients were included. The overall point prevalence of patients with at least one HAI was 10.6%. The most frequent types of infections were pneumonia (34.3%), bloodstream infections (10.5%), systemic infections and urinary tract infections (10.5% and 9.1%, respectively). In 14 (12.4%) cases, the pathogen responsible for HAI was SARS-CoV-2 following onsite spread, accounting for almost 10% of all HAIs. Microorganisms were identified in 60.1% of HAIs. Antimicrobials were administered in 711 (59.8%) patients, with 1.59 antimicrobials used per patient. Conclusion: The prevalence of HAI and antimicrobial use among hospitalized patients in Crete, Greece was similar to the national HAI prevalence in 2016 despite the enormous pressure on public hospitals due to the COVID-19 pandemic. Nevertheless, both HAI prevalence and antimicrobial use remain high, underlining the need to implement adequate infection control and antimicrobial stewardship interventions.