We conducted a study exploring the potential of the medicinal plant Ziziphus spina-christi as a source of antimicrobial compounds. With the growing threat of antibiotic resistance, we were particularly interested in evaluating plant-derived natural products for their ability to fight infections. We collected Z. spina-christi leaves from four different regions in Sudan and extracted them with ethanol. Our phytochemical screening revealed the presence of several secondary metabolites, including flavonoids, triterpenes, sterols, tannins, saponins, cardiac glycosides, and reducing sugars. Alkaloids and anthraquinones were not detected. We then tested the ethanolic extracts for antimicrobial activity against a range of bacterial and fungal pathogens using the agar well diffusion method. Interestingly, we observed significant differences between the extracts from different locations. The extract from Khartoum city exhibited the highest antimicrobial potency, followed by the extracts from Al-Rahad, Al-Hidiba, and finally Al Damazin. Based on these findings, we concluded that Z. spina-christi leaves contain a diverse array of phytochemicals with potential medicinal applications. The variation in antimicrobial effects between the extracts may be attributed to environmental factors influencing the plant's chemical composition. Further research is warranted to identify the specific bioactive compounds responsible for the observed antimicrobial activities. This could pave the way for the development of plant-based antimicrobial agents to combat drug-resistant pathogens and alleviate the global public health crisis posed by the rise of antibiotic resistance.