Endophytes are fungi that colonize the internal tissues of plants without causing immediate adverse effects. Saudi Arabia (SA) is rich in Opuntia ficus-indica cacti and the cactus-like plant Aloe vera, which grow in the southern and western areas of SA. This study aimed to isolate and identify endophytic fungi from cacti and cactus-like plants in the Jeddah, Taif, and Al Baha regions KSA and then determine their effects on pathogenic fungal and bacterial growth. The isolates were groupedAloe Vera; Opuntia Ficus-Indica; Endophytic Fungi; Antimicrobial Activities; Pathogenic Bacteria. into 16 distinct operational taxonomic units based on the sequence of the internal transcribed spacer in the rDNA gene with the primers ITS1 and ITS4. Mucor circinelloides was the endophytic fungus found most frequently, with a relative frequency of 20.43%, followed by Talaromyces funiculosus, with a relative frequency of 16.12% when isolated from Opuntia ficus-indica and Aloe vera. Nine out of sixteen endophytic fungi exhibited strong antifungal activity against all the tested pathogens. P. funiculosum, Aspergillus versicolor, Penicillium janthinellum, and Fusarium oxysporum showed vigorous antimicrobial activities against the human pathogenic bacteria Escherichia coli, Shigella sp., and Salmonella typhimurium.