Following a series of published analyses ranging from architecture to prehistoric diet of the Iron Gates’ inhabitants, our research aims to present new results regarding use-wear analysis of knapped stone artefacts from Lepenski Vir and Padina with a particular focus on the transition from Mesolithic to Neolithic in this region. Use-wear and residue analysis are applied using the low and high-power approach by Optical Light Microscope (OLM) observations combined with FTIR analysis. Based on the results, some of the main activities that took place in Iron Gates are processing of hide, bone, antler, plants, and soft stone. It is very important to highlight the complexity of various processes, which make this already specific area more peculiar. A variety of both simple, but overall, more complex and composite activities are recorded with the elaborate preparation of the used materials, for example, hide. Particular processes, such as butchering, were noted both inside the houses, and also concentrated in precise, specific areas of the settlements, where only tools involved in the processing of hide and meat were found. The data obtained highlight the activities of these advanced hunter-gatherer-fishermen and first farmers communities. Together with spatial analysis, the dynamics and processes in the Late Mesolithic and Early Neolithic are revealed, but also many questions regarding the specialization of the prehistoric settlements on the Danube are posed.