Background: It is becoming more widely acknowledged that Pseudomonas aeruginosa (P. aeruginosa) is a significant nosocomial pathogen that can cause serious infections. In general, new therapeutic approaches that prioritize the utilization of extracts and physiologically active chemicals extracted from herbal plants should be used to control P. aeruginosa infections. The World Health Organization (WHO) estimates that in underdeveloped countries, around 80% of the population mostly uses traditional medicine and plant extracts as their primary source of medicine to treat a variety of infectious diseases. Objective: The purpose of this study was to investigate the in vitro antimicrobial activity of A. vera against P. aeruginosa isolated in Khartoum state hospitals. Materials and Methods: The present descriptive study was performed in Khartoum state. Between November 2022 and April 2023, a total of 100 clinical isolates of P. aeruginosa were obtained from patients hospitalized at Khartoum State hospitals. Standard microbiological techniques, such as the Gram stain, catalase test, and oxidase test, were used to identify the isolates. Ciprofloxacin, Ceftriaxone, Meropenem, and Gentamicin. A. vera gel were used in an antimicrobial susceptibility test against the P. aeruginosa strains. Results: Isolates of P. aeruginosa were collected from hospitalized individuals. Of the 100 isolates included in the study, 63 isolates (63%) had been isolated from males and 37 isolates (37%) had been isolated from females. Using the microdilution method, antimicrobial susceptibility testing for 100 P. aeruginosa clinical isolates was conducted for four antibiotics. The results showed that 38% of the isolates were resistant to ciprofloxacin, 94% to ceftriaxone, 82% to gentamicin, and 0% to meropenem. Conclusions: P. aeruginosa demonstrated multidrug resistance to a widely used antibiotic. A different therapeutic agent for the treatment of clinical multi-drug resistance bacteria may be employed in the ethanol extracts of Aloe vera since they have shown antibacterial efficacy against multi-drug resistant P. aeruginosa which is resistant to many drugs and may be treated with a combination of conventional antibacterial discs and the crude extract of Aloe vera. The study's findings validate the traditional wisdom that aloe vera plants may cure microbial infections and suggest that aloe vera may be used to make powerful new antibacterial compounds.