This study was conducted to investigate the antagonistic activity of endophytic and rhizosphere fungi isolated from a medicinal plant, Sesuvium portulacastrum, against P. aphanidermatum, the cause of damping-off of cucumber. A total of 41 endophytic and 19 rhizosphere fungi were isolated from S. portulacastrum. Except one Basidiomycota isolate, all the other isolated fungi belonged to the phylum Ascomycota. Five of the isolates gave above 50% suppression of P. aphanidermatum during in vitro dual culture tests. Molecular and Phylogenetic analysis identified all antagonists as Aspergillus insulicola A435, A. insulicola A419, and A. melleus A412 and two rhizosphere isolates; A. terreus A213 and A. luchuensis A116. Scanning electron microscopic studies indicated deleterious effects such as hyphae wall damage and abnormal mycelial growths of Pythium mycelia, which were induced by all antagonists. Except for isolate A116, the culture filtrates of the other antagonists significantly increased the electrolyte leakage from Pythium mycelia; whereas ethyl acetate extracts of antagonists A435, A412, and A213 showed significant growth suppression. GC-MS analysis of metabolites identified a range of antimicrobial, antifungal and plant growth promoting volatile compounds such as fatty acids, alcohols, alkenes, ketones and triterpenes. All the five antagonists were able to produce varying amounts of hydrolytic enzymes, such as- cellulase and β-glucanase. However, antagonist A435, A412 and A213 showed significantly higher cellulase (0.3-02 filter paper activity/ml) activity. Antagonist A435 and A116 showed the highest β-glucanase activity (≤ 46 units/ml). Controlled glasshouse growth experiments supported the findings of in vitro tests and enzyme assays where A435 and A116 showed up to 70 % control of damping-off, with 50% increased survival than controls. The isolates A412 and A213 showed 57 % and 52 % seedling survival with up to 30- 40 % damping-off control. The lowest was A419 with only 20 % higher seedling survival than the control. The antagonists A435, A116, and A213 also contributed to increased cucumber shoot length, as well as shoot and root dry mass of cucumber. The synergetic effects of metabolites and hydrolytic enzymes could be the reason for the variation between isolates in the antagonistic activity and cucumber growth promotion. The study identified A. insulicola A435, A. terreus A213 and A. luchuensis A116 as potential biocontrol agents P. aphanidermatum causing damping-off of cucumber. The ability of these isolates to inhibit other soil-borne pathogens should be investigated.