The bacterial isolates obtained from the botanical garden soil of the National Research Center in Dokki, Giza, Egypt, showed high levels of cyclodextrin glycosyltransferase (CGTase) activity. The CGTase superior Bacillus (WT3) was classified as Bacillus hunanensis using the 16S-ribosomal RNA sequence and the Basic Local Alignment Search Tool (BLAST). The mutagen ethyl methane sulfonate (EMS) was used to create CGTase mutants of WT3 that produced more. After the testing of 30 mutants chosen in a screening assay, the best mutant for CGTase biosynthesis was WT3-60-8, which produced 84.19 U/mL with a 110.6% relationship to the parental-strain. Protoplast fusions between WT3-60-4 and WT3-60-8 mutants were carried out to increase CGTase activity. Twenty-seven fusants were collected, with fusant (F1-8) showing a 122.4 percent improvement in CGTase activity over its parental strain. Many different DNA banding patterns were observed while fingerprinting with random amplified polymorphic DNA (RAPD). Furthermore, the genetic backgrounds of the two excellent mutants and four superior fusants, as well as the parental-strain, were divided into three clusters, the phylogenetic tree was obtained, and the genetic backgrounds of the two excellent mutants and four superior fusants, as well as the parental-strain, were detected based on genetic distances.