Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (FON), is a predominant and devastating soil-borne disease that results in significant yield losses in watermelon cultivation. In this study, a strain MM isolated from the herbage rhizosphere soil, exhibited an inhibition rate of 65.46% against FON, leading to mycelial collapse, atrophy, and deformation. In pot experiments, strain MM effectively controlled Fusarium wilt of watermelon, showing a control efficacy of 74.07%. Through morphological observation and 16S rDNA gene sequencing, strain MM was identified as Serratia plymuthica. Additionally, S. plymuthica MM demonstrated antagonistic activity against eight plant pathogens, indicating that MM had broad-spectrum antifungal activity. The strain also exhibited the ability to synthesize siderophores and indole acetic acid (IAA), both of which are growthpromoting compounds. Moreover, strain MM secreted various extracellular enzymes, including protease, chitinase, -glucanase, and cellulase. This ability allowed S. plymuthica MM to readily colonize watermelon roots and promote seedling growth. Inoculation with S. plymuthicaMMincreased the activity of PAL, POD, PPO, and CAT enzymes associated with watermelon defense. Furthermore, qRT-PCR analysis revealed up-regulation of LOX, POD, PAL, ClPR3, and C4H genes, which are related to plant disease resistance. The results indicated that S. plymuthica MM enhances watermelon plants’ resistance to FON by activating the JA, SA, and shikimic acid phenylpropanoid–lignin synthesis pathways. Gas chromatography–mass spectrometry (GC-MS) analysis of S. plymuthica MM culture supernatant identified piperazinedione, pyrrolo[1,2-a]pyrazine-1,4-dione, and octadecenamide as the main antimicrobial substances. Overall, S. plymuthica MM shows promise as a biocontrol agent against Fusarium wilt of watermelon, suggesting its potential for the development of a new biocontrol agent.