The global population growth has led to a higher demand for food production, necessitating improvements in agricultural productivity. However, abiotic and biotic stresses pose significant challenges, reducing crop yields and impacting economic and social welfare. Drought, in particular, severely constrains agriculture, resulting in unproductive soil, reduced farmland, and jeopardized food security. Recently, the role of cyanobacteria from soil biocrusts in rehabilitating degraded land has gained attention due to their ability to enhance soil fertility and prevent erosion. The present study focused onNostoc calcicolaBOT1, an aquatic, diazotrophic cyanobacterial strain collected from an agricultural field at Banaras Hindu University, Varanasi, India. The aim was to investigate the effects of different dehydration treatments, specifically air drying (AD) and desiccator drying (DD) at various time intervals, on the physicochemical properties ofN. calcicolaBOT1. The impact of dehydration was assessed by analyzing the photosynthetic efficiency, pigments, biomolecules (carbohydrates, lipids, proteins, osmoprotectants), stress biomarkers, and non-enzymatic antioxidants. Furthermore, an analysis of the metabolic profiles of 96-hour DD and control mats was conducted using UHPLC-HRMS. Notably, there was a significant decrease in amino acid levels, while phenolic content, fatty acids, and lipids increased. These changes in metabolic activity during dehydration highlighted the presence of metabolite pools that contribute to the physiological and biochemical adjustments ofN. calcicolaBOT1, mitigating the impact of dehydration to some extent. Overall, present study demonstrated the accumulation of biochemical and non-enzymatic antioxidants in dehydrated mats, which could be utilized to stabilize unfavorable environmental conditions. Additionally, the strainN. calcicolaBOT1 holds promise as a biofertilizer for semi-arid regions.