In this study, biochar was produced from three biomass feedstocks such as fruits of Cassia fistula and Caesalpinia sp. and barks of Eucalyptus globulus. The samples of the obtained biochar were characterized for pH, physiochemical properties, surface morphology, and surface functional groups. The obtained biochar samples were further studied with/without the combination of urea for their plant growth enhancement properties including the germination studies and effect on shoot and root growth of rice plants. Biochar produced from C. fistula fruits at 1.5% concentration increased the plant shoot height 18% higher than the control plants. Eucalyptus sp. barks’ biochar application at 0.5% concentration also increased the plant shoot height 12% longer than the control. However, the biochar produced from Caesalpinia sp. did not increase the shoot length. Yet, a generalized increase in root length was observed with the application of biochar. The combined application of nitrogen fertilizer (urea) and biochar together reverted the effect of biochar on the shoot length increase. Phospholipid-derived fatty acid (PLFA) characterization of soil revealed that soil biota shifts when soil was supplemented with the biochar. The bacterial community increased and a loss of fungal community was observed with the application of biochar.