This study aimed to explore the fermentative performance of nine lactic acid bacterial strains with probiotic potential during sorghum fermentation. The strain’s attributes including proliferation counts, pH levels, production of organic acid antibacterial activity, and their ability to break down starch were evaluated during the fermentation period in the presence and absence of glucose as a carbon source. In addition, the inhibitory activity of these potential probiotic strains against pathogenic bacteria (Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus) was examined through a co-culturing technique. The results demonstrated that all 4 Lactobacillus strains exhibited robust growth in both glucose and glucose-free fermentation experiments. Glucose supplementation significantly enhanced lactic acid yield which ranged from 0.19 to 0.44% compared to fermentation without glucose which ranged from 0.04 to 0.29%. The selected Lactobacillus strains effectively lowered the media pH below 4.0 after 24 h, producing substantial lactic acid. Notably, in the absence of glucose, only Lb. helveticus D7 and Lb. amylolyticus D12 achieved pH levels below 4 after 8 h, producing the highest lactic acid amounts of 0.27 and 0.29% after 24 h, respectively. Amylase activity was detected on two strains, D7 and D12. Furthermore, most of the tested Lactobacillus strains demonstrated complete inhibition (6 log to 0 Log CFU/mL) of pathogen growth after 24 h of co-culturing, suggesting their potential for enhancing the safety quality of sorghum-based fermented products.