The objective of this study is to evaluate the bacterial contamination of smartphones of healthcare workers (HCWs), and to estimate the potential risks of transmission of antibiotic-resistant pathogens between patients and HCWs. During the campaign day for hand cleaning awareness, 107 swabs were collected from the screens of personal smartphones belonging to HCWs of the Integrated University Hospital of Verona. All the samples were cultured and grown colonies identified using the mass spectrometry technology Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF MS, Biomerieux^®). All smartphones tested were colonized by at least one bacterial genus. Twenty-two different genera were isolated. The majority of isolates were Bacillus species, accounting for 71.96%, coagulase negative staphylococci (CoNS), accounting for 57.01%, other Gram-Positive bacteria (22.43%), and Gram-Negative bacteria (3.74%). Pathogenic bacteria were Aerococcus viridans (4.17%), Klebsiella pneumoniae (5.26%), Pseudomonas spp. (21.05%), Escherichia coli (10.53%), Enterococcus spp. (20.83%), Acinetobacter spp. (42.11%) and Staphylococcus aureus (14.02%). All strains of Enterococcus spp. did not show any antibiotic resistance. S. aureus were Methicillin-sensitive. Unfortunately, K. pneumoniae and A. baumannii were resistant to carbapenems. Results showed that HCWs’ smartphones were contaminated with different types of bacteria. The personal and daily usage of disinfectant wipes and hand disinfectant gel may help to reduce the risk of transmission and contamination of nosocomial infections in hospitals.