Investigations into natural resource use and access are often limited to rural areas; such use is not considered an integral part of urban livelihood strategies, especially amongst the poor. With growing urban food insecurity, poverty, and unequal access to services, natural resource use may provide a viable alternative to cash-based resources, thus, allowing households to navigate the rural–urban continuum as they address livelihood needs. This may be especially pertinent for growing, but small, urban landscapes that are in transition. We investigated the use of and access to natural resources in households in rural (Lesoma), peri-urban (Kazungula), and urban (Kasane) settlements in northern Botswana. Semi-structured questionnaires for 85 households were used to investigate household demographics, variety of natural resources used, the availability, use, and importance of natural resources, threats to resources, harvesting locations, and extent of resource commercialization. Significant differences were identified in the mean number of resources used by households across the three settlements (Kruskal–Wallis Chi-squared = 9.29, df = 2, p = 0.01). Using the post hoc test Conover with Bonferroni adjustment, mean natural resource use between the urban and peri-urban villages did not differ significantly (p = 1). However, both types of villages differed from the rural village in mean resource use (urban p = 0.007 and peri-urban p = 0.012). Nevertheless, urban and peri-urban households reported use of a broad range of natural resources, highlighting the importance of these products in transitioning landscapes. Across the study villages, natural resource harvesting occurred predominantly on communal land. Primary barriers to resource access were perceived to be strict government regulations and decreasing resource availability. Natural resource commercialization was identified as a potential opportunity but was often carried out only on a small scale. The use of natural resources is intuitively thought to be associated with rural areas, but our results suggest that such resources form an important part of livelihoods across urban and peri-urban landscapes as well. This continued reliance on natural resources raises important planning questions about how to ensure both the ongoing conservation of forested and other natural areas, and the availability of associated resources for urban livelihoods. In this regard, small urban towns that are rapidly transitioning from rural landscapes provide a targeted opportunity for early intervention. Our findings underpin the vital role that natural areas play in supporting the livelihoods of the urban poor and highlight the need to encourage land designation and management of such areas not only for conservation but also as a safety net for vulnerable urban households.