This paper investigated the impact of the combined use of spent mushroom substrate (SMS) biochar and plant-growth-promoting rhizobia (PGPR) on the growth, yield, and biochemical response of cauliflower (Brassica oleracea var. botrytis). A preliminary study was conducted under greenhouse condition using six treatments (sextuplicate) as control (no addition), T1 (PGPR), T2 (5 g/Kg biochar), T3 (5 g/Kg biochar + PGPR), T4 (10 g/Kg biochar), and T5 (10 g/Kg biochar + PGPR) under greenhouse conditions. The Scanning Electron Microscopy (SEM-Zeiss), Energy Dispersive Spectroscopy (EDS), and Fourier’s transform infrared spectroscopy (FTIR) analyses showed that biochar produced from slow pyrolysis of SMS had advantageous structural, functional, and morphological properties for agricultural use. Results showed that SMS biochar addition aids the acceleration of soil nutrient properties. SMS biochar and PGPR application also significantly (p < 0.05) improved the selected growth, yield, and biochemical parameters of cauliflower. In particular, the highest cauliflower yield (550.11 ± 10.05 g), fresh plant biomass (1.66 ± 0.04 Kg), dry plant biomass (149.40 ± 4.18 g), plant height (22.09 ± 0.14 cm), root length (11.20 ± 0.05 cm), plant spread (28.35 ± 0.18 cm), and the number of leaves (12.50 ± 0.50) were observed in T5 treatment. Similarly, the best values for biochemical parameters and enzyme activities such as total chlorophyll (TC: 3.13 ± 0.07 mg/g), superoxide dismutase (SOD: 79.12 ± 1.29 µg/g), catalase (CAT: 55.70 ± 2.52 µg/g), peroxidase (POD 30.18 ± 0.37 µg/g), total phenolics (TP: 19.50 ± 0.31 mg/g), ascorbic acid (AA: 14.18 ± 0.55 mg/g), and total carotenoids (TCT: 150.17 ± 8.20 µg/100 g) were also recorded in the T5 treatment. The application of SMS biochar and PGPR showed a positive correlation with growth, yield, and biochemical response of cauliflower, as indicated by the Pearson correlation analysis. The findings of this study suggest efficient recycling of mushroom industry waste for biochar production and the use of PGPR to improve nutrient utilization in sustainable agriculture.