Objectives: Subcritical water extraction technique is considered as an environmentally extraction technique. The aim of this study was to compare the different characteristics of water extract and subcritical water extract of Helicteres isora L. 

Materials and Methods: Water extraction was performed under the following conditions: 25°C, 24 h, and solid-to-water ratio 1:30. Subcritical water extract was carried out under specific conditions (pressure = 10 bar, temperature = 160°C, solid-to-water ratio = 1: 30, time = 30 min). Chemical composition analysis was performed using GC–Mass chromatography. Anti-biofilm activity in the terms of anti-attach and removal of biofilm were assessed using the ELISA reader method and reading absorbance at 570  nm. Anti-microbial activity against Bacillus cereus, Staphylococcus aureus, Staphylococcus saprophyticus, and Bacillus subtilis was investigated by measurement of inhibitory zone diameter. Anti-enzymatic and antioxidant properties were also assessed. 

Results: The results of GC–Mass analysis showed some components extracted in subcritical method which were absent in water extract such as octadecanoic acid, hexadecanoic acid, and berberin. Antioxidant activity of the two tested extracts revealed that subcritical water extract had more antioxidant capacity than water extract (P ≤ 0.05). The two tested extracts exhibited anti-enzymatic activity against polyphenol oxidase enzyme with better performance of subcritical water extract. Anti-biofilm activity of the two extracts implies that, in the case of preventing biofilm formation, both extracts had similar efficiency but in the removal of biofilm, subcritical water extract showed better performance. Both extracts had anti-microbial activity against B. cereus, S. aureus, S. saprophyticus, and B. subtilis with better performance of subcritical water extract. Anti-enzymatic assay also showed similar results. 

Conclusions: Subcritical water extract of H. isora showed more antioxidant activity as well as antibiofilm, anti-bacterial, and anti-enzymatic activity rather than ordinary water extract.