This study presents a comprehensive evaluation of environmental susceptibility associated with the development of the Belgrade-Novi Sad Railway Route in Serbia. Fostering socio-economic growth through transportation infrastructure necessitates an acute awareness of potential ecological implications, a nexus often overlooked in the haste of progress. Accordingly, a broad spectrum of environmental parameters such as land use, air and noise pollution, water resources, and biodiversity were systematically assessed. The employment of Habitat Equivalency Analysis facilitated the discernment of potential environmental detriments linked with the railway project. Significant findings from this investigation offer critical insights for policymakers, urban planners, and environmental conservationists, thus enriching the understanding of ecological consequences attached to the expansion of the specified railway corridor. These findings, serving as a tool for informed decision-making, are pivotal in striving towards a balanced approach between the exigencies of transportation infrastructure enhancement and the indispensable goal of environmental preservation. Ultimately, the goal of this study is to promote an enhanced understanding of the reciprocal relationship between infrastructure development and environmental impact, thereby contributing to the ongoing discourse on sustainable practices beneficial for current and future generations.