Melanization of shrimp gills is a general symptom in shrimp black bill diseases (sBG); therefore, the cause is not immediately apparent. This study aimed to determine the primary and secondary agents associated with a black gill outbreak in the Delvar complex in the Bushehr Province of Iran. During July and August of 2020, 450 healthy and affected shrimps were collected based on brown to black gill color from three farms of the Bushehr Province in Iran. Characterization of pathogens was performed by gross signs, wet mounts, microbiological tests, histopathology methods, and sequencing of molecular markers. The mean weight and length of infected shrimps was significantly (P < 0.05) lower than the healthy shrimps. The histological examination of sBG indicated the adhesion of secondary lamellae, accumulation of hemocyte cells at the end of the secondary lamellae, and necrosis of epithelial cells with pyknosis and karyorrhexis. According to our results, Aspergillus sydowii as a primary agent correlates with sBG disease in Litopenaeus vannamei from Iran, and other biotic factors, including bacterial (Vibrio owensii and V. parahaemolyticus) probably are secondary agents associated with sBG disease.