Introduction: The efficacy of Javanese turmeric (Curcuma xanthorrhiza Roxb.) extract in inhibiting the growth of C. albicans biofilm decreases as the biofilm gets mature. phospholipase and proteinase are C. albicans hydrolytic enzymes which facilitate the adhesion and invasion of the fungi into the host cell. Objective: Analyzing the activity of phospholipase and proteinase enzymes on three phases of C. albicans biofilm whose growth has been inhibitted by Javanese turmeric ethanol extract. Methods: After 1,5 hour incubation, the C. albicans biofilm in the treatment group were exposed to Javanese turmeric ethanol extract at its Minimum Biofilm Inhibitory Concentration (MBIC50) which was determined by MTT assay, the negative controls were exposed to nothing, while the positive controls were exposed to Nystatin. C. albicans biofilm were further incubated for 6, 24, and 48 hours to achieve initial, intermediate, and maturation phases. Activity of the hydrolytic enzymes were analyzed by measuring the diameter of precipitation/ proteolytic zone that were seen surrounding the colony zone of C. albicans on Egg yolk Agar (for observing Phospholipase activity) and Bovine Serum Albumin agar (for observing Proteinase activity). Results: In early, intermediate, and mature phases C. albicans biofilm which had been inhibited by Javanese turmeric ethanol extract, both the diameters of precipitation / proteolytic zone of phospholipase and proteinase were smaller compared to negative controls. Conclusion: Inhibition effect of Javanese turmeric ethanol extract toward the growth of three phases of C. albicans biofilm could decrease the activity of phospholipase and proteinase enzymes of C. albicans. © 2024 Universiti Putra Malaysia Press. All rights reserved.