The aim of this study was to determine the effects of cultivar, nitrogen (N) rate, and biostimulant application on the chemical composition of the aboveground biomass of perennial ryegrass (Lolium perenne L.). A small-area field experiment was established in the Agricultural Experiment Station in Tomaszkowo (53°42′40.8″ N 20°26′04.7″ E, north-eastern Poland). The experiment had a split-plot design with three replications, and the experimental variables were as follows: (i) perennial ryegrass cultivar: Bajka and Baronka, (ii) N fertilizer rate: 0, 120, 240 kg N ha−1, (iii) application of biostimulants: Blatt Boden-Foliar (BB-F) and Blatt Boden-Foliar + Blatt Boden-Multical (BB-F + BB-M). This study demonstrated that the tetraploid cultivar Baronka had a more desirable chemical composition than the diploid cultivar Bajka. The biomass of cv. Baronka had a higher content of CP, CF, and K, and it was characterized by higher leaf greenness (SPAD) values. Nitrogen fertilization considerably increased the content of CP, P, and K, and leaf greenness (SPAD) values in both cultivars, and the noted increase was higher when N was applied at 240 kg ha−1. The N fertilizer rate of 120 kg ha−1 led to a significant decrease in the average Ca content of plants, whereas the N fertilizer rate of 240 kg ha−1 had no significant effect on Ca concentration. The tested biostimulants significantly affected the chemical composition of perennial ryegrass biomass, and their influence was greater when they were applied in combination. The foliar application of Blatt Boden-Foliar and Blatt Boden-Multical increased the content of CP, P, and chlorophyll in perennial ryegrass leaves, whereas it decreased the accumulation of CF, K, and Ca in plants. The analyzed biostimulants had a positive effect on the chemical composition of perennial ryegrass biomass. This is an important practical consideration because high-quality green fodder for livestock can be produced while minimizing the use of mineral fertilizers and adverse environmental impacts.