Background: In the underdeveloped world, microbial infections constitute a serious public health concern. About half of all deaths in tropical nations are thought to be caused by infectious diseases. In addition, some well-known pathogens have assumed new epidemiologic importance by acquiring antimicrobial resistance. Some plants are known as medicinal because they contain active substances that cause specific reactions relating to the cure of human disease. Acacia nilotica, commonly known in Sudan as Garad or Sunt, has many medicinal uses. Objectives: This study used the agar diffusion method to study the antibacterial activity of the different concentrations of ethanolic extract of the medicinal plant Acacia nilotica pods against selected gram-negative bacteria. Methodology: A cross-sectional study was done in Shendi Town, River Nile State, at the microbiology laboratory at Shendi University. Eighty-five wound swabs and urine specimens were collected from patients with symptoms of urinary tract infection and wound infection who attended Shendi Teaching Hospital and Almek Nimir University Hospital from February to March 2023; bacteria were isolated and identified using colonial morphology, Gram stain, and biochemical tests. Using the agar well diffusion method, we tested the Acacia nilotica extract at 100%, 50%, 25%, and 12.5% w/v concentrations. Results: This study found infection was persistent in patients aged 26–34. Escherichia coli was a primary isolate. According to gender, out of 85 specimens, 26 (30.6%) were males, whereas 59 (69.4%) were females. The ethanolic extract of Acacia nilotica pods was screened for their antimicrobial activity against clinical isolates of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, Enterobacter, Citrobacter, Serratia, Providencia, and Proteus vulgaris. The antimicrobial effect of the extract was compared with the standard drugs. Crude extracts of Acacia nilotica inhibited the growth of various bacteria and showed its broad spectrum antimicrobial potential, which may be employed in managing microbial infections. Conclusion: The fruits of Acacia nilotica were the subject of these investigations, demonstrating its potent in vitro activity against clinically isolated bacteria and its potential as a treatment for various bacterial and viral infections.