The pervasive expansion of human-engineered infrastructure, particularly roads, has fundamentally reshaped landscapes, profoundly affecting wildlife interactions. Wildlife-vehicle collisions, a common consequence of this intricate interplay, frequently result in fatalities, extending their detrimental impact within Protected Areas (PAs). Among the faunal groups most susceptible to road mortality, reptiles and amphibians stand at the forefront, highlighting the urgent need for global comprehensive mitigation strategies. In Greece, where road infrastructure expansion has encroached upon a significant portion of the nation’s PAs, the plight of these road-vulnerable species demands immediate attention. To address this critical issue, we present a multifaceted and holistic approach to investigating and assessing the complex phenomenon of herpetofauna road mortality within the unique ecological context of the Lake Karla plain, a rehabilitated wetland complex within a PA. To unravel the intricacies of herpetofauna road mortality in the Lake Karla plain, we conducted a comprehensive 12-year investigation from 2008 to 2019. Employing a combination of statistical modeling and spatial analysis techniques, we aimed to identify the species most susceptible to these encounters, their temporal and seasonal variations, and the ecological determinants of their roadkill patterns. We documented a total of 340 roadkill incidents involving 14 herpetofauna species in the Lake Karla’s plain, with reptiles, particularly snakes, being more susceptible, accounting for over 60% of roadkill occurrences. Moreover, we found that environmental and road-related factors play a crucial role in influencing roadkill incidents, while spatial analysis techniques, including Kernel Density Estimation, the Getis-Ord Gi*, and the Kernel Density Estimation plus methods revealed critical areas, particularly in the south-eastern region of Lake Karla’s plain, offering guidance for targeted interventions to address both individual and collective risks associated with roadkill incidents.