Abstract: Background and Objective: At the beginning of planting dragon fruit, seedlings often have decay at the base of the stem caused by the fungus Sclerotium rolfsii (S. rolfsii) Sacc. This disease is very detrimental for dragon fruit farmers. An alternative control of this disease may also use biological antagonistic microbes. The aim of this research was to evaluate Pseudomonas fluorescens (P. fluorescens) P60 against Sclerotium rolfsii in dragon fruit seedlings and its plant growth-promoting ability. Methodology: Experiments were conducted for the dragon fruit plantations of Agrowisata Kusumo Wanadri at Glagah Village, Temon Subdistrict, KulonProgo Regency. There were five treatments which includes: 1: Untreated control, 2: P. fluorescens P60 as a drench, 3: P. fluorescens P60 as a soak, 4: P. fluorescens P60 as a soak and drench and 5: Benomyl as a soak and drench. There were six replicates of each treatment and arranged in a randomized complete block design. The soak and drench were repeated six times, each for 10 min, 30 days after planting. Following the treatments, the following parameters were evaluated such as disease incidence, late populations of S. rolfsii, plant fresh weight, root length, number of shoots, the shoot length and qualitative phenolic compound contents. Data were analyzed by F-test to determine the effect of treatment. Results: This study results showed that the use of P. fluorescens P60 effectively suppress stem rot caused by S. rolfsii on dragon fruit seedlings, by inhibiting the incubation period as 29.76%, lower the disease intensity as 66.65%, suppress the disease incidence of 46.14%, lower late sclerotia population of 74.79% and increase the content of phenolic compounds qualitatively. The application of P. fluorescens P60 was most appropriate to the nursery dragon fruit cuttings material by soaking in a solution of P. fluorescens P60 for 10 min. Conclusion: The use of P. fluorescens P60 can promote the growth of dragon fruit seedlings, covering the difference in seedlings fresh weight of 52.87%, root length as 40.09%, number of shoots as 34.76% and long shoots as 8.99%.