Background: Intensive care unit (ICU) infection management is a growing challenge, and physicians should have regularly updated antibiograms. The aim of this study was to find out the prevalence of pathogens and to determine their antibiotic susceptibility in different ICUs of an Egyptian tertiary care hospital. This retrospective record-based cross-sectional study was conducted from the first of January to the last of December 2019 with a total of 45,221 diagnostic first-isolate culture/patient obtained from different ICUs in Zagazig University Hospitals. The antibiogram construction was done according to Clinical and Laboratory Standards Institute instructions and a Web-based antibiogram at Stanford University. Results: The positive blood isolate was the most prevalent infection site (32.37%) followed by sputum and urine isolates. Gram-negative microorganisms (74.41%) were the most common pathogens, with Klebsiella pneumoniae as the most frequently identified one with an incidence of 33.51% followed by Escherichia coli with 19.3% incidence. Antibiotic sensitivity showed that colistin is the most effective antibiotic with 96.2%, 94.7%, and 89.9% sensitivity for Klebsiella, E. coli, and Acinetobacter, respectively, while carbepenems sensitivity was extremely low, showing 19.5% and 19% imipenem and meropenem sensitivity for Klebsiella, 48% imipenem and 52.7% meropenem sensitivity for E. coli, 20.1% imipenem and 20.3% meropenem sensitivity for Acinetobacter, and 17.3% imipenem and 15.2% meropenem sensitivity for Pseudomonas aeruginosa. Fungal infection in our results represented less than 1%. Conclusion: Our study provides a local baseline epidemiological data which describes the extent of the ICU infections problem in this tertiary care hospital. Trial registration: ClinicalTrials.gov (NCT04318613) Keywords: Intensive care unit, Microorganisms, Antimicrobial resistance, Antibiogram