Tomato fruit rot severely impacts yield and quality, causing economic losses. This study aimed to identify the pathogenic fungi associated with post-harvest tomato fruit rot and characterize the transcriptomic responses of tomatoes. Pathogens were isolated from diseased tomato fruit tissues and identified using morphology, phylogenetic analysis, and in vitro pathogenicity tests. The genome of Cladosporium oxysporum Co-1 was assembled and annotated. RNA-seq analysis was used to profile transcriptional changes in tomatoes infected with C. oxysporum Co-1, with RT-qPCR validating the RNA-seq data and spectrophotometric assays analyzing the host physiological responses. Three pathogenic fungi were isolated. Colonies of C. oxysporum exhibited a near-circular shape, with colonies transitioning from an olive-green center to gray-green at the edges, and based on ITS, β-tubulin, and EF-1α gene sequences, this isolate exhibited 99% identity with C. oxysporum. The other two fungal isolates were identified as Alternaria alternata and Fusarium incarnatum, respectively, based on morphological and multi-locus sequence analysis. All three strains induced fruit rot and browning in tomatoes, confirming their pathogenicity. The genome size of C. oxysporum Co-1 was 34,515,558 bp, comprising 52 scaffolds with a GC content of 52.82%, and encoding 10,081 protein-coding genes. RNA-seq analysis showed dynamic gene expression changes in tomatoes infected with strain A, with differentially expressed genes enriched in pathogenicity-related pathways. Spectrophotometric assays revealed that peroxidase and superoxide dismutase activities decreased initially followed by an increase post-inoculation with C. oxysporum, indicating that tomatoes defend against pathogen infection through the antioxidant enzyme system. These findings revealed the pathogenic fungi were associated with post-harvest tomato rot disease, provided genomic resources for C. oxysporum, and provided insight into the host’s response to this strain.