Jalapeño peppers (Capsicum annuum var. Jalapeño) represent one of the most important crops in Mexico. However, many plant diseases, such as wilt caused by strains of the genus Fusarium, reduce its yield. A sustainable alternative to control diseases is the use of biological control agents (BCAs), for example, beneficial microorganisms such as strains of the genus Bacillus. This study aims to analyze the potential use of B. cabrialesii subsp. tritici TSO2T as a BCA and elucidate its potential modes of action against Fusarium strains causing wilt in Jalapeño peppers. For this, symptomatic samples were collected in a commercial field in the Yaqui Valley, Mexico. Six Fusarium isolates were morphologically and molecularly characterized. After pathogenicity tests, F. languescens CE2 was found to be pathogenic. In screening assays for biocontrol bacteria, strain TSO2T, which was isolated from soil in a wheat commercial field under an organic production system and preserved in the Culture Collection of Native Soil and Endophytic Microorganisms (COLMENA), had the best biocontrol effect against CE2, and its cell-free filtrate reduced mycelial growth by 30.95%. Genome mining (antiSMASH) of strain TSO2T allows us to identify gene clusters associated with biocontrol, such as fengycin, surfactin, bacillibactin, bacilysin, bacillaene, subtilosin A, and sporulation killing factor, which codify to antimicrobial metabolites and are associated with swarming motility of the studied beneficial strain. These results demonstrate the effectiveness of B. cabrialesii subsp. tritici TSO2T as a potential BCA for the control of Fusarium wilt, through competition and a complex of antifungal metabolites, which still need to be validated through metabolomic analysis. This study highlights the importance of the use of integrative genomic and bioactivity-guided methodologies in understanding biocontrol pathosystems.