The pyrolysis of biomass material results in pyroligneous acid (PA) and biochar, among other by-products. In agriculture, PA is recognized as an antimicrobial agent, bio-insecticide, and bio-herbicide due to antioxidant activity provided by a variety of constituent materials. Application of PA to crop plants and soil can result in growth promotion, improved soil health, and reduced reliance on polluting chemical crop inputs. More detailed information regarding chemical compound content within PA and identification of optimal chemical profiles for growth promotion in different crop species is essential for application to yield effective results. Additionally, biochar and PA are often applied in tandem for increased agricultural benefits, but little is known regarding the optimal proportion of each crop input. This work reports on the effect of combined applications of different proportions of PA (200- and 800-fold dilutions) and chemical fertilizer rates (100%, 75%, 50%, and 0%) in the presence or absence of biochar on Komatsuna (Brassica rapa var. perviridis, Japanese mustard spinach) plant growth. To elucidate the chemical composition of the applied PA, four different spectroscopic measurements of fluorescence excitation were utilized for analysis—excitation-emission matrix, ion chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. It was determined that PA originating from pyrolysis of Japanese pine wood contained different classes of biostimulants (e.g., tryptophan, humic acid, and fulvic acid), and application to Komatsuna plants resulted in increased growth when applied alone, and in different combinations with the other two inputs. Additionally, application of biochar and PA at the higher dilution rate increased leaf accumulation of nutrients, calcium, and phosphorus. These effects reveal that PA and biochar are promising materials for sustainable crop production.