Microhabitat characteristics can be used as a proxy to predict the community structure of associated organisms and evaluate their vulnerability to habitat degradation. Microhabitat-specific and ectothermic taxa (like many reptiles) are among the best models to study responses to changing habitats and climate. We examined the niche breadth and guild structure of reptiles from Agasthyamalai Hills in the southern Western Ghats of India based on microhabitat use data. We recorded a total of 47 reptile species from 1,554 observations comprising two major orders and 11 families. Niche breadth analysis revealed that 45% of reptiles are microhabitat specialists, indicating the importance of protecting their habitats with all structural attributes. Cluster analysis grouped reptile species into four major guilds based on microhabitat preferences. The forest floor-dwelling guild was the largest group with 25 species, followed by the semi-arboreal guild with 12 species. The floor-dwelling guild also exhibited both the highest number of microhabitat specialists (n = 11) and globally threatened species (n = 3), highlighting the need for preserving ground cover characteristics such as leaf litter, boulders, and open ground for conserving reptiles in the region. Considering the microhabitat specializations within the community, we recommend a dynamic approach to monitor abundance, diversity, and habitat quality across the Agasthyamalai landscape to better conserve its rich reptile diversity.