The objective of this study was to determine how plant species, fertilizer potential acidity/basicity rating (PABR), and fertilizer concentration affect root substrate pH. Three experiments were conducted. In the first experiment, 13 herbaceous species were grown in a root substrate of three sphagnum peatmoss: one perlite (v/v) with deionized water and a neutral fertilizer (NF) with a PABR of 0 for 78 days to determine species relationships to substrate pH. The decrease in substrate pH ranged from 0.14 to 2.45 units, depending on species. In the second experiment, four of the 13 species from the previous trial representing the range of pH suppression were grown under similar growth conditions as the first experiment for 70 days. Substrate pH was lowered in the range of 0.47 to 2.72 units. In the third experiment, three fertilizers with PABRs of 150 kg·t−1 CaCO3 equivalent alkalinity, 0 neutral, and 193 kg·t−1 CaCO3 equivalent acidity were applied in a factorial design at 100 and 200 mg·L−1 N at each irrigation to kalanchoe (the species with the greatest pH suppression from the previous experiments) for 56 days. When applied at the lower fertilizer rate (100 mg·L−1 N), the PABRs resulted in the final substrate pH levels of 4.68, 5.60, and 6.11 for the acidic fertilizer (AF), NF, and basic fertilizer (BF), respectively. At the high fertilizer rate (200 mg·L−1 N), substrate pH declined continuously to 3.97, 4.03, and 4.92 for the AF, NF, and BF, respectively. Expression of PABR depended on the balance between the abiotic (chemical) effect of the fertilizers vs. the biotic (physiological) effects of the fertilizers on microbes and plants. The PABR was best expressed when the fertilizer supply was just adequate or lower indicating a closer connection to the biotic effect.