This study aimed to isolate and identify the pathogens accompanying the rotting and death of cotton seeds and seedlings. Also, some Trichoderma spp. were assessed against the fungal pathogen associated with the disease. The results showed that one of the essential isolated fungi, Alternaria alternata, has demonstrated high virulence in attacking cotton seeds and seedlings and reducing germination and growth. This fungus was identified based on its morphological and molecular characteristics. The Trichoderma species applied have shown high efficiency in reducing infection rates and increasing cotton germination percentage. Every isolate of Trichoderma showed a high efficiency against the fungus A. alternata by providing the highest antagonistic ability, reaching 93.75%. The highest percentage of inhibition growth of the pathogen (86.11%) was achieved by Trichoderma koningiopsis, while the lowest percentage of inhibition growth of the pathogen was 66.65 % for Trichoderma reesei. However, the biological formula prepared from species Trichoderma viride, Trichoderma pseudokoningii, Trichoderma koningiopsis and Trichoderma reesei displayed the highest percentage of inhibition of 100% against the fungus A. alternata. Keywords. Alternaria alternata; Trichoderma spp.; Trichodermin; gliotoxin; Biological control.