The fate of antibiotics has mostly been studied in lab-scale systems. Few studies have precisely evaluated the attenuation of antibiotics in natural streams. We used Lagrangian sampling combined with a tracer test and one-dimensional transport with inflow and storage model (OTIS) to reveal the effects of dilution and self-attenuation processes, and diurnal variation of light on the degradation of 14 antibiotics in a 3.6 km low-flow stream. The results showed that the order of in-stream attenuation rates were macrolides (0.18–0.25 h−1) > tetracyclines (0.16–0.18 h−1) > fluoroquinolones (0.094–0.13 h−1) > sulfonamides (0.056–0.082 h−1); half of the mass of antibiotics were lost within 0.44–1.96 km. The dilution effect, including longitudinal dispersion and transient storage, accounted for 20.70%–91.60% of the total attenuation while self-attenuation processes accounted for 8.40%–79.30%. Over 60% of sulfonamides were dissipated by dilution, while over 68% of the removal of macrolides and tetracyclines was attributed to self-attenuation. A comparison of the attenuation rates between day and night demonstrated that photo-dependent attenuation played a dominant role, especially for sulfonamides, accounting for more than 50% of their self-attenuation. Photo-independent attenuation reduced most macrolides and tetracyclines. This in situ experiment increased our understanding of antibiotic attenuation in natural streams.