This dataset includes files associated with a 21-day nutrient diffusing substrate experiment run in Pear Lake of Sequoia National Park, California in summer 2022. The experiment was designed to evaluate the effects of different treatment levels of a macronutrient (nitrogen) and a micronutrient (copper) on periphyton growth and community structure in an alpine lake. While the effects of nitrogen on primary productivity are well-studied, including in remote mountain lakes, the potential effects of low concentrations of necessary micronutrients are poorly understood. This experiment used three nitrogen treatments (0, 0.05, 2 mg/L NO3-N) and six copper treatments (0, 0.005, 0.05, 0.5, 1, 5 mg/L Cu), totaling 18 unique treatment combinations. For each treatment, we collected three replicate samples for periphyton copper concentrations, periphyton growth as ash-free dry mass, and periphyton community structure quantified using metabarcoding methods. We used a 16S rDNA primer to quantify bacteria taxa, including cyanobacteria, and an 18S rDNA primer to quantify eukaryotic primary producers. The sequencing data (fastq files) are stored in a separate repository with the National Center for Biotechnology Information (NCBI; BioProject PRJNA1200755). The remaining data are published in this release, including the periphyton copper concentrations (NDS_copper.csv) and ash-free dry mass data (NDS_afdm.csv). Additionally, this data release includes the data from our lab test to verify that copper would diffuse from agar (labDiffusionTest.csv), metadata associated with each of the field experiment’s sample units (NDS_treatment.csv), the water quality results collected at the start and end of the experiment (NDS_watqual.csv), and a file that includes a list of all of the Phyla identified in the samples via metabarcoding, the count of each Phyla, and the broad category that we assigned to the Phyla for downstream analyses (phylaDescriptions.csv). Water chemistry data collected with passive data loggers (pH, dissolved oxygen, conductivity, and temperature) are also associated with this experiment, but those data are released in a separate data release (https://doi.org/10.5066/P1T6UYAX; hourlyLoggers.csv and hourlyLoggers.rds).