Periphytic algae attached to a streambed substrate (periphyton) are an important primary producer in stream ecosystems. We determined natural radiocarbon abundances in chlorophyll a (Δ¹⁴C_chl) and bulk (Δ¹⁴C_bulk) of periphyton collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Periphyton Δ¹⁴C_chl values (–258‰ in April and –190‰ in October) were slightly lower than Δ¹⁴C_bulk values (–228‰ in April and –179‰ in October), but were close to the Δ¹⁴C value for dissolved inorganic carbon (DIC) (–217 ± 31‰), which is a mixture of weathered carbonates (Δ¹⁴C = –1000‰), CO₂ derived from aquatic and terrestrial organic matters (variable Δ¹⁴C) and dissolved atmospheric CO₂ (Δ¹⁴C approximately +30‰ in 2013). Although the bulk isotopic composition of periphyton is recognized as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89% to 95% algal carbon (derived from ¹⁴C-depleted DIC) and 5% to 11% terrestrial organic carbon (derived from ¹⁴C-enriched atmospheric CO₂).