Long-term use of chemical fertilizers is affecting the environment, soil quality, and biodiversity. Organic agriculture is gaining global attention by using microbial-based biofertilizers. Carriers protect microbes by providing nutrition, energy, and suitable conditions for their survival while entering the natural environments. The purpose of this study was to evaluate the ability of different carrier materials to enhance the yield and the quality of spinach and to select the best carrier material for spinach biofertilizer. Three pre-isolated and characterized bacterial strains (AN-35, ZM-27, and ZM-63) were tested for their compatibility and used in this experiment through seed inoculation with organic carriers, i.e., compost, peat, press mud, biochar, and charcoal. A pot experiment and a field experiment were conducted to evaluate the efficacy of different organic carriers. The results of the pot study showed a significant increase in spinach growth, i.e., shoot length (25%), shoot fresh weight (24%), root length (25%), and root fresh weight (29%), spinach nutrition, i.e., nitrogen (18%), phosphorus (22%), potassium (15%), iron (17%), and zinc (14%), spinach physiology, i.e., relative water content (27%), chlorophyll content (9%), and the membrane stability index (28%) under peat coated treatments with 24% more soil microbial populations compared to the control. Similarly, in the field experiment, peat coating significantly enhanced spinach growth, i.e., shoot length (29%), shoot fresh weight (23%), root length (16%), and root fresh weight (24.7%), spinach nutrition, i.e., nitrogen (16%), phosphorus (19%), potassium (15%), iron (17%), and zinc (23%), spinach physiology, i.e., relative water content (28%), chlorophyll content (13%) and the membrane stability index (32%), and spinach yield per hectare (30%), as well as producing 20% higher soil microbial populations. From these results, it is concluded that peat is a good carrier material for biofertilizer production as it not only enhances crop production but also the microbial number, in addition to improving soil quality.