Benchalokawichian (BLW) remedy as a Thai Traditional medicine preparation, consist of five plant roots as follows: Ficus racemosa Linn., Capparis micracantha DC., Clerodendrum petasites S. Moore., Harrisonia perforata Merr., Tiliacora triandra Diels.. Water extract and ethanolic extracts of individual plants and Benchalokawichain remedy were tested for in vitro cytotoxic activity against three lung cancers (A549, COR-L23, H226), oropharynx and larynx cancer cell lines (KB, Hep2). All of the water extracts showed no cytotoxic activity (IC50>50 μg/mL). Four of ethanolic extracts which were FR, CM, CP, HP also showed the same result. The ethanolic extract of Tiliacora triandra (TTE) inhibited the growth of all types of respiratory system (three lung cancer, oropharynx and larynx) cancer cell lines with IC50 values in range of 19.5- 45.2 μg/mL. In the same way, the 95%EtOH extract of Benchalokawichien (BLWE) showed in vitro cytotoxic activity against A549, COR-L23, H226, KB, Hep2 cell lines with IC50 values in the range of 10.1-33.7 μg/mL. Both of them showed specific cytotoxicity against H226 compared with other cell lines. In addition, BLWE showed higher cytotoxicity against the skin cancer (C32) cell line than TTE with IC50 values of 29.1 and 37.9 μg/mL, respectively.The results of this research suggest that the Tiliacora triandra ethanolic extract (TTE) would be a good marker for cytotoxic activity of BLW remedy, so it was investigated using a bioassay-guided fractionation technique. Following VLC method, five fractions (TTF1-TTF5) were collected, a percentage of yield was calculated. TTF4 was the most abundant compound (48.4%) followed by TTF5, TTF2, TTF3, TTF1 (24.1%, 4.6%, 3.82%, 0.1%). The result showed that TTF4 like TTE exhibited in vitro cytotoxic activity against all cancer cell lines. Surprisingly, TTF5 showed specific against H226 with an IC50 value of 33.02 μg/mL. TTE1 was isolated from the TTF4 ethanolic extract of Tiliacora triandra. This is first report of tiliacoric acid [4-((3S,5R)-5-decyl-4-oxo-1,2-dioxolan-3-yl) butanoic acid] a new compound whose structure was elucidated by spectroscopy (NMR, MS, IR, UV). CPE1 compound which was isolated from the Clerodendrum petisites ethanolic extract was identified as oleanolic acid acetate. The main compound of Harrisonia perforata was HPE1, identified as perforatic acid. Compound BLWE1-3 was isolated from the chloroform fraction of crude BLW ethanolic extract. By comparison of 1H and 13C NMR data with previous studies, BLWE1-BLWE3 were identified as β-sitosterol, pectolinarigenin and perforatin A or Omethylalloptaeroxylin, respectively.Cytotoxic activity of isolated compounds against two lung cancer cell lines, melanoma, and a normal cell line were investigated. The TTE1 inhibited the growth of two lung cancer cell lines, A549 and H226, with IC50 values of 6.49 and 13.98 μg/mL, which was better than TTE (IC50=33.65 and 19.48 μg/mL). Notably, TTE1 showed no toxicity in a normal cell line (HaCaT). CPE1 also showed strong cytotoxic activity against A549 and H226 with IC50 values of 1.24 and 1.95 μg/mL whereas CPE wasn’t active (>50 μg/mL). Although HPE1 was the main compound from Harrisonia perforata and Benchalokawichian remedy, it showed no cytotoxic activity in SRB assay (>50 μg/mL). In contrast, BLWE2 was cytotoxic to two lung cancer cell lines (A549, H226) and melanoma cancer cell line (C32) with IC50 values of 7.76, 6.25, and 6.78 μg/mL respectively. However, BLWE2 also showed toxicity in a normal cell line. From literature reviews, pectolinarigenin and α-mangostin were used as the markers of mixed extract (Benchalokawichian or BLW and Garcinia mangostana or GM) in our research. Comparison of GM yield in various solvent extractions, the ethanolic extract showed the highest yield 21.38%, following soxhlet extraction with methanol, chloroform and hexane were 20.82%, 4.35%, 0.92%, respectively. The results showed that BLWE inhibited S. aureus MRSA and S. aureus at a concentration 5 mg/mL, but it wasn’t killing them at the same dose (MBC >5mg/ml). In similarly, BLWE exhibited antimicrobial activity against S. pyogenes at a concentration 400 μg/mL (MBC>400μg/mL). In an investigation of the antimicrobial activities against S. aureus MRSA,GMC showed the highest activity follow by GMH, GME, MIX 1:2 and MIX 1:1 (MIC values 0.78, 1.56, 3.13, 6.25, 12.5 μg/mL, respectively). Alpha-mangostin exhibited higher antibacterial activity against MRSA than vancomycin (MIC=0.19, 0.78 μg/mL, MBC=0.39, 0.78 μg/mL, respectively) while gentamicin and clindamycin were not active (MIC >100 μg/mL). The GMH extract showed highest anti-inflammatory activity, followed by GMC (IC50 values 6.24 , 7.84 μg/mL). Nevertheless, they also showed toxicity at a concentration of 30 μg/mL. Both MIX1:1 and GMM showed moderate antiinflammatiory activity (IC50 = 34.70, 37.84 μg/mL), and they showed no toxicity at a concentration of 50 μg/mL. It is possible that BLWE can reduce toxicity of GME. ...