This study aims to investigate the effects of the combined treatment of biochar (BC) and arbuscular mycorrhizal fungi (AMF) on cotton plants, focusing on morphological and physiological characteristics, nutrient content, and soil enzyme activities. The study examined the effects of different biochar doses in mycorrhiza-inoculated (M +) and non-inoculated (M -) treatments (0% BC + 200 kg N ha(-1), 1.5% BC + 200 kg N ha(-1), 3% BC + 200 kg N ha(-1)). The results showed that the highest SPAD value (34.66%) was observed in the M( +) + 3.0% BC treatments. NDVI values increased by 25.29% in M( +) + 3.0% BC treatments. M( +) treatment increased the N, NO3-, and soil enzyme activity was higher in the M( +) treatment. However, enzyme activity decreased as BC dose increased in both M( +) and M( -) treatments, with the most significant decrease observed in the 3.0% BC treatments. Nitrate reductase enzyme activity (NRA) decreased in all treatments except the 3.0% biochar treatment. M( +) treatment increased soil urease content by 27.24% compared to the M( -) treatment. Additionally, plant height, root length, nodule count, and spore number increased with increasing biochar doses, with the highest increase observed in the M( +) + 3.0% BC treatments. Positive correlations were found between SPAD, NDVI, and nutrients such as Fe, Cu, Mn, and root attributes. Negative correlations were observed with Mg and CAT enzyme activity. Urease, DHG, and CAT enzyme activities decreased significantly with increasing BC doses, particularly in M( +) treatments. Biochar and AMF co-treatment enhanced growth, root parameters, spore count, NDVI, SPAD, and soil and plant enzyme activities in cotton plants. The combined treatment of biochar and AMF is beneficial for cotton production. The findings of this research have significant potential implications for sustainable cotton production and soil management practices, particularly in optimizing mycorrhiza and biochar to enhance soil fertility, improve water retention, and reduce the need for chemical fertilizers.