Due to its structure and properties, diatomite from a deposit in Jawornik Ruski (Subcarpathian Voivodeship) can be used as a sorbent in rain gardens. The purpose of the current research is to analyze how enriching the substrate used in a rain garden with diatomite can affect the removal of biogenic pollutants. This study was carried out under laboratory conditions using retention columns, two experimental columns with different contents of diatomite, and a control column without the addition of diatomite. Analyses of the materials used included studies of the characteristics of the rain garden layers (water permeability and granulometric analysis) and characterization of the diatomite (SEM images, oxide and phase composition, leachability, and BET). The effects of diatomite on pollutant removal were studied for NH4+, PO43−, NO3−. The results showed approximately 3-fold higher reductions in the concentration of NH4+ and PO43− in the columns with the addition of diatomite than in the control one (reduction in the concentration of NH4+ by 93 and 94% and of PO43− by 94 and 98% with the addition of 20 and 30% diatomite contents, respectively). The study results confirmed the possibility of removing contaminants using diatomite, thus reducing their entry into the aquatic environment.