Integrated approach to planning, building, maintaining and monitoring the health of transportation system and mountain ecosystem needs to be addressed in the Himalayan State of Uttarakhand. An ambitious mega transportation project, costing US $171.43 billion has been launched by Government of India on December 27, 2016. The project envisages and upgradation of the exiting 889 km of five highways leading to the four major shrines of India. A large scale of rock cutting, road widening and the construction of 15 big bridges, 101 small bridges, 3,596 culverts and 12 by passess under the mega project are being taken up in Uttarakhand (Latitude 29ᴼ26ʹ-31ᴼ28ʹN; Longitude 77ᴼ49ʹ-80ᴼ06ʹE). The study on the stretch of 140 km of NH 58 (Rishikesh to Rudraprayag) was undertaken for a period of two-years (January 2017 to December 2018). Springs are three-way ecotone where groundwater, surface water and terrestrial ecosystem interact. Despite the unique characteristics along with high biodiversity, no comprehensive documentation of the impact of transportation on the crenic habitats and biodiversity has been addressed considerably. A total of 23 major crenic habitats (helocrene, limnocrene and rheocrene) have been identified on NH-58. A drastic change in the geomorphology and the values of turbidity, conductivity, dissolved oxygen, alkalinity, total dissolved solid, coarse particulate organic matter (CPOM) and fine particulate organic matter (FPOM) has been recorded in crenic habitats. Crenobiodiversity of the springs represented by periphyton, macrophytes, zooplankton and macrozoobenthos, has been affected adversely due to the construction of this project. Most affected were the periphyton and macrozoobenthos dwelling the crenic habitats. A decline of 39.78% in annual mean density, and 46.9% in Shannon Weiner Index of periphyton was recorded during two-year period. Periphyton were represented by genera of Bacillariophyceae (14), Chlorophyceae (11) and Cynophyceae (4). A depletion of 43.8% in annual mean density, and 32% in Shannon Weiner diversity index of macrozoobanthos was computed during the period of study. Macrozoobenthos of crenic habitats were represented by the genera of Ephemeroptera (12), Plecoptera (05), Trichoptera (03), Diptera (05), Coleoptera (04), Odonata (03), Mollusca (03) and Annelida (03).Regression analysis and Canonical Correspondence Analysis (CCA) was performed between environmental variables and dominant taxa of crenobionts for assessing the impacts. Mitigation measures - regular monitoring of biotic components, spatial-temporal variables of spring flow, delineation of the spring recharge basin, protection of associated spring brooks, stabilizing spring banks, by protecting riparian vegetation, removal of barriers between groundwater, spring and spring brooks have been recommended. Further rock cutting and widening of road should be done after the GIS geomapping of the crenic habitats to be affected by the project.