Periphyton and water were sampled 1 km upstream of the Cookeville Wastewater Treatment Plant (PR_upstream), 2 km downstream of the discharge (PR_downstream), and from a tributary (PO) 10 km south of the wastewater treatment plant. Percent composition of 113 algal taxa were documented. Assemblages of soft-bodied algae and diatoms at the PR_downstream site had more eutrophic taxa than assemblages at the other sites. Water at the PR_downstream site had >7-fold higher concentration of total phosphorus (193 µg·L^-1), yet <1.5-fold higher concentration of total nitrogen (1900 µg·L^-1) than water at the other sites. The total nitrogen to total phosphorous ratio (TN:TP ratio) at the PR_downstream site (9.8) was below the value hypothesized for N-limitation of algae growth (10), whereas the ratios at the PR_upstream site (65.9) and PO site (62.4) were above. Phosphorus and nitrogen additions to in vitro growth assays using Raphidocelis subcapitata indicate in vitro carrying capacity for Raphidocelis subcapitata was P limited in water from the PR_upstream and PO sites. In vitro carrying capacity for Raphidocelis subcapitata was significantly greater and N-limited in water from the PR_downstream site. The results indicate that reduction of the TN:TP ratio of Pigeon Roost Creek changed the nutrient that limits in vitro carrying capacity for R. subcapitata from P to N, and imply that standard growth assays using Raphidocelis subcapitata to evaluate carrying capacity of P-enriched stream water may not indicate the extent of the impact of the P-enrichment to a watershed due to N-limitation of carrying capacity in vitro.