The impact of antibiotic residue on sediment ecology at the watershed level is not yet fully understood. In this investigation, varying concentrations of oxytetracycline (OTC) and sulfadiazine (SD) were added to the overlying water of both the upper (0–10 cm) and bottom sediment (20–30 cm) layers at the watershed scale to evaluate the ecological impact on sediment habitats through the analysis of the activities of enzymes, namely urease (UA), alkaline phosphatase (APA), peroxidase (POA), and dehydrogenase (DHA). Results showed that the levels of UA and APA in the bottom sediment layers exceeded those in the top sediment layer upon reintroduction of antibiotics. Conversely, the fluctuations in DHA were notably reduced across various types of antibiotics and exposure concentrations in the bottom sediment layers. Within the top sediment layers, as the concentration of OTC exposure increased, there was a corresponding elevation in POA levels. However, the response of POA initially ascended and subsequently descended with rising SD exposure concentration, although it consistently exceeded the control levels. In contrast, the response of DHA displayed an inverse correlation with OTC exposure concentration but a direct correlation with SD exposure concentration. At the watershed scale, under antibiotic exposure, UA and DHA exhibited significantly higher levels upstream compared to downstream. Conversely, APA and POA appeared relatively stable across the watershed following the reintroduction of antibiotics. Moreover, DHA demonstrated a noticeable decreasing trend with increasing concentrations of OTC exposure. Environmental factors had a predominant influence, exceeding 40%, on enzyme activities during antibiotic reintroduction. Specifically, particle size significantly inhibited enzyme activity, while sediment nutrient conditions, including total carbon, nitrogen, and sulfur content, significantly enhanced enzyme activities. The study suggests that enzyme activities associated with antibiotic reintroduction in watershed sediments are established during stable stages in the bottom sediment layer or downstream sediment environment as part of sedimentary and transport processes. More research is required to explore the maintenance and evolution of antibiotic resistance profiles in the presence of long-term antibiotic residues.