While it is well acknowledged that both light irradiance and biofilm age influence daytime nutrient cycling in streams, it remains unclear how these factors interact and affect nighttime nutrient dynamics together with dissolved organic matter (DOM) composition. The understanding of these interactions is crucial for comprehending overall nutrient dynamics in stream ecosystems. In this study, we assess the interplay of biofilm age (one, i.e. younger, and three, i.e. older, weeks old) under three levels of light irradiance (high, low, and no light) on the daytime and nighttime dynamics of dissolved inorganic nitrogen (NO3–N and NH4–N), soluble reactive phosphorus (SRP), and DOM molecular fractions in streamside flumes. Daytime NO3–N demand by younger biofilms increased with irradiance, with no net-uptake without light. Moreover, both daytime and nighttime NO3–N net-uptake increased with biofilm age under higher light incidence, but at lower rates for nighttime net-uptake. Older biofilms acted as daytime sources of DOM (humic-like molecular fractions) and of SRP, while protein-like DOM fractions were consumed both during daytime and nighttime by both younger and older biofilms. Our results reveal distinct daytime and nighttime nutrient dynamics influenced by light irradiance and biofilm age, emphasizing the importance of nighttime processes for a comprehensive assessment of nutrient cycling in streams.