Streams and rivers are widely impacted by human activities ranging from hydrological modifications to point and non-point pollution. Among the pollutants that enter urban lotic ecosystems are pharmaceuticals and personal care products, including antibiotics, that may play a role in the occurrence of antibiotic resistance genes (ARGs). Regardless of the factors that determine abundance of ARGs in urban streams, the public health and ecological impacts of ARGs may be profound but are largely understudied. Approaches to examination of ARGs are varied and key knowledge must be obtained to develop management strategies. Tinker’s Creek (NE Ohio) was used as a model urban stream to assess ARGs by collection of invertebrates, water and sediment from seven locations with varying land use and water quality. Microbiome communities were distinct across time and location and ARGs were more abundant at more urban locations during particular times of the year. ARG differences extended to stream macroinvertebrates with ARGs tending to have higher abundance in their guts than the surrounding environment. However, laboratory experiments in microcosms using two invertebrates from the study stream (Corbicula and crayfish) did not reveal that macroinvertebrates were vectors of spread of ARGs. Overall, factors that appear to be drivers of ARGs, such as composition of the microbiome, environmental conditions, and metal resistance genes, were related to the extent of urban land use directly or indirectly. Implications of the work include management strategies focused on sources (including wastewater treatment) and impact on humans (including persistence of antibiotics after drinking water treatment) as well as emerging strategies to examine ARGs and their importance to ecosystem and human health.