Data are needed to assess the relationships between urbanization and biodiversity to establish conservation priorities. However, many of these relationships are difficult to fully assess using traditional research methods. To address this gap and evaluate new acoustic sensors and associated data, we conducted a multimethod analysis of biodiversity in a rapidly urbanizing county: Greenville, South Carolina, USA. We conducted audio recordings at 25 points along a development gradient. At the same locations, we used refugia tubes, visual assessments, and an online database. Analysis focused on species identification of both audio and visual data at each point along the trail to determine relationships between both herpetofauna and acoustic indices (as proxies for biodiversity) and environmental gradient of land use and land cover. Our analysis suggests the use of a multitude of different sampling methods to be conducive to the completion of a more comprehensive occupancy measure. Moving forward, this research protocol can potentially be useful in the establishment of more effective wildlife occupancy indices using acoustic sensors to move toward future conservation policies and efforts concerning urbanization, forest fragmentation, and biodiversity in natural, particularly forested, ecosystems.