Background and objectives. Contracting infectious diseases from microbial contaminated door handles is a potential threat to health and safety. Therefore, we performed microbiological screening of door handles at the door handles of Zawia Education Hospital, Zawia, Libya, to estimate the presence of potentially pathogenic organisms. Methodology. A total of 125 samples were collected aseptically by using a moistened sterile bacteriologic swab on different days by scrubbing the selected handles and immersing them in the sterile broth to be transported to the microbiology laboratory, within no more than 30 minutes after collection. All the bacterial isolates were characterized and identified by standard diagnostic microbiological procedures and criteria, according to the Clinical and Laboratory Standards Institute. Data were presented as counts and percentages using Excel spread data sheath. Results: There was a total of 147 isolates obtained from the 12 sampled departments. The frequency of isolation of these organisms from these samples was as follows: coagulase-negative staphylococcus (16.80%) in 21 samples, Staphylococcus aureus (10.40%) in 13 samples, Diphtheroid (76.00%) in 95 samples, non-lactose fermenting gram negative bacilli (12.80%) in 16 samples, streptococcus pyogenes (0.80%) in one sample, and Klebsiella pneumoniae (0.80%) in one sample. Conclusion. Staphylococcus aureus, coagulase negative staphylococcus, streptococcus pyogenes, klebsiella pneumonae, Diphtheroid, and non-lactose fermenting gram-negative bacilli were the main bacteria isolated in this research study. The results of the present study demonstrated that contact surfaces such as door handles are often colonized by several bacteria and serve as a potential source of infections.