Nowadays thermal springs are worldwide known sources of valuable Biobricks for biotechnological applications. The ecological analysis is a prerequisite to exploring thermophiles cyanobacteria that are a pervasive group in thermal springs globally. Therefore, this study aims to ecologically analyze cyanobacteria in Al-Lith area to extract growth factors that control prokaryotic primary producers and species biodiversity. Previously, there are four known thermal spring in Al-Lith, Saudi Arabia. This study discovered additional six novel springs that were never mentioned before. A seasonal sampling of cyanobacteria and physicochemical factors carried out from winter, 2015 to spring of 2016 in order to analyze factors controlling cyanobacteria species diversity. Temperature, total dissolved salt (TDS), pH, and dissolved oxygen and 17 chemical elements were investigated. A total of 34 species were identified in Al-Lith springs. Ghomygah thermal spring is the largest spring in Al-Lith region that is rich in high diversity of cyanobacteria. Oscillatoria and Chroococcus are dominant species and functionally tuned to all thermal habitat in Al-Lith springs regardless of the different growth conditions among them. The multivariate analysis helps resolve the complex relationships between physicochemical parameters and cyanobacteria biodiversity. Temperature is the pre-eminent factor determine the distribution of mat-forming cyanobacteria in spite of the difference in other factors. The remarkable increase in pH was observed with increases in cyanobacteria coverage. Therefore, pH is directly controlled by cyanobacteria. Thermal springs in Al-Lith grouped primarily based on physicochemical characteristics and secondly based on their geographical location. This work provides a comprehensive characterization of thermophiles native habitat that formulate a foundation for biotechnological deployment of cyanobacteria. Indeed, rational conservation of operative and healthy ecosystems are imperative to preserve these valuable resources.