Bacterial keratitis is a potentially blinding corneal infection, withandbeing the leading gram-positive and gram-negative pathogens, respectively. The increasing antimicrobial resistance among these pathogens necessitates the development of newer therapeutic approaches. This study aims to assess the therapeutic efficacy of mesenchymal stem cell-derived conditioned medium (MSC-CM) for ocular isolates ofand. The MSCs were derived from human adipose tissue, bone marrow, dental pulp, and umbilical cord. The antibacterial effects of MSC-CM were studied through zone of inhibition and colony-forming unit assays, whereas morphological changes were studied through scanning electron microscopy. Furthermore, to mimic bacterial infections, human corneal epithelial cells were stimulated with bacterial endotoxins, and changes in the expression of antimicrobial peptides (LL-37, Human Beta Defensin-3 [HBD-3], Dermcidin, Hepcidin-25, and Lipocalin-2) and inflammatory mediators (IL-6 and TNF-α) were evaluated through enzyme-linked immunosorbent assay. While HBD-3 was present in the highest concentration across all sources of MSCs, treatment with MSC-CM further upregulated the expression of antimicrobial peptides while downregulating the expression of pro-inflammatory mediators, suggesting its immunomodulatory effects. The results suggest that MSC-CM exhibits antibacterial effects against the tested ATCC strains and clinical isolates ofandcausing ocular infections, with the highest efficacy shown by the adipose tissue-derived MSC-CM. Furthermore, in anhuman corneal infection model, adipose-derived MSC-CM decreased the bacterial burden while preserving corneal transparency compared to untreated controls. These findings indicate the promising therapeutic potential of MSC-CM for ocular bacterial infections.