Background: Mycetoma is a unique neglected tropical disease caused by a substantial number of microorganisms of fungal or bacterial origins. Identification of the causative organism and the disease extension are the first steps in the management of the affected patients and predicting disease treatment outcome and prognosis. Objectives: To determine the role of special stain in identification of fungi in eumycetoma among Sudanese patients at Soba University Hospital. Method: This descriptive cross- sectional study was conducted of Fifty-nine archival formalin fixed paraffin- embedded tissue blocks, four identical sections from each archival paraffin-embedded tissue block were obtained and stained with routine H&E and special stains {Periodic-Acid-Schiff (PAS) and Grocott-Gomori silver (GMS) and Gridley stain}, then the collected data was analyzed using SPSS version 23.0. Results: The use of special stains especially periodic acid Schiff's and Grocott methenamine-silver (GMS) stain are of value (specific and sensitive) in identifying the fungal hyphae and yeast of eumycetoma. Mycetoma is more commonly reported in males than females; also mycetoma is most common in young adults (16–40 years old) and is uncommon in children. No significant relationship was demonstrated between diagnosis and variables (gender, age). Type I and II was the commonest type of the host tissue reaction encountered in this study. Conclusion: Results showed that GMS and PAS had the ability to stain both fungal hyphae and yeast in compare to Gridley's stain which had some limitations to stain fungal structures.