To develop hydroethanolic extract of leaves of Psidium guajava L. (Myrtaceae), identify novel target and lead compound for pediatric non alcoholic fatty liver disease (ped-NAFLD). Hydroethanolic extract (PG-HM) and sequential fractions of locally collected leaves of P.guajava were prepared and standardized for quercetin content using LC-MS/MS. The effect of PG-HM on markers of ped-NAFLD in weaned rats with unrestricted access to liquid fructose (15%) till puberty and early adulthood, was evaluated. The PG-HM was investigated using 1) HepG2 cells grown in fructose containing medium for intercellular signalling, 2) isolated murine hepatocytes for mitochondrial effects, 3) jejunum isolated from fructose drinking rats for effect on ex vivo fructose transport, 4) molecular dynamics for in silico binding of quercetin with GLUT5, 5) normal rats for oral pharmacokinetics. The quercetin content in PG-HM was 3630.66 ± 17.61 ng ml-1 and the Cmax and Tmax were 140.527 ± 5.718 ng ml-1 and 0.75 ± 0.079 h, respectively. The PG-HM mitigated ped-NAFLD through hepatic signalling pathways of 1) leptin-insulin (Akt/FOX-O1/SREBP-1c), 2) hypoxia-inflammation (HIF-1ɑ/VEGF, TNF-ɑ), 3) mitochondrial function (complexes I-V), 4) oxidative stress (MDA, GSH, SOD) and 5) glycolysis/gluconeogenesis/de novo lipogenesis (hexokinase, phosphofructokinase, ketohexokinase, aldehyde dehydrogenase). For the first time, the PG-HM is reported to reduce and delay the fructose transport across jejunum that may be implicated to the binding of quercetin with GLUT5. For prevention and treatment of ped-NAFLD, the PG-HM, GLUT5 and quercetin are evidenced as dietary supplement, drug target and lead compound, respectively.