Non-alcoholic fatty liver disease (NAFLD) is a problem in obese people caused by increasing intake of high-calorie food such as fructose implicated in the elevated prevalence. It is necessary to identify novel drugs to develop effective therapies. In this study, we combined LOPAC® (The Library of Pharmacologically Active Compounds) and High-Content screening to identify compounds that significantly reduced intracellular lipid droplets (LD) after high fat medium (HFM) treatment. Among 1280 compounds, we identified 239 compounds that reduced LD by >50%. Of these, 17 maintained cell viability. Nine of them were selected for validation using normal primary hepatocytes, of which five compounds showed dose-dependent efficacy. Whole genome transcriptomic network analysis was performed to construct the underlying regulatory network. There were 831 (711 up-regulated and 120 down-regulated genes) and 3480 (2009 up-regulated and 1471 down-regulated genes) genes that showed a significant change (>2-fold; p < 0.05) after 12 and 24 h HFM treatment, respectively. Gene enrichment and pathway analysis showed several immune responses mediated by MIF, IL-17, TLR, and IL-6. These compounds modulate lipogenesis via GSK3β and CREB1, which is followed by an alteration in the expression of several downstream genes related to hepatocellular carcinoma and hepatitis. CREB1 is a core transcription factor and may be a potential therapeutic target for liver disease. In conclusion, this proof of concept provides a strategy for identifying novel drugs for treatment of fatty liver disease as well as elucidates their underlying mechanisms. This research provides opportunity for developing future pharmaceutical therapeutics.