Introduction:In order to minimize chance of amputation due to actinomycetoma, it is important to correctly identify the causative agents. Microscopic examination of grains is not definite and further confirmatory diagnostic tests are needed. This study aims to investigate the prevalence of actinomycetoma and to explore the usefulness of strb1 gene in the diagnosis of the disease. Materials & Methods: The present study is a prospective cross-section laboratory-based study in which clinical samples (n = 100) from patients with mycetoma lesions were collected. The samples were cultured on Lowenstein Jensen and glucose yeast extract agar media. Grown colonies were initially identified using Gram's stain, Ziehl Neelsen stain, and selected biochemical reactions. Confirmation was done by the analysis of polymerase chain reaction amplified strb1 gene. Results: Actinomycetoma was represented by a high ratio (12%) among the study population. Nine out of the 12 isolates (75%) were found to belong to the genus Streptomyces; whereas three isolates (25%) were identified as Nocardia spp. on the basis of phenotypic and mycolic acid contents. Conclusion: It could be concluded that actinomycetoma exists with significant prevalence (12%) among patients investigated in the present study. Streptomyces is the most important etiological agent of actinomycetoma compared to Nocardia.