Overexpression of pyruvate dehydrogenase kinases (PDKs) has been widely noticed in a variety of human solid tumors, which could be regarded as an attractive therapeutic target for cancer therapy. In this paper, we present an enzymatic screening assay and multiple biological evaluations for the identification of potential PDKs, especially PDK1 inhibitors. We identified 9 potential PDKs inhibitors from the screening of an in-house small molecule library, all of the identified inhibitors reduced pyruvate dehydrogenase (PDH) complex phosphorylation. Among which, 4, 5, and 9 displayed the most potent PDKs inhibitory activities, with EC<sub>50</sub> values of 0.34, 1.4, and 1.6 μM in an enzymatic assay, respectively. A kinase inhibition assay suggested that 4, 5, and 9 were pan-isoform PDK inhibitors, but more sensitive to PDK1. Meanwhile, the three compounds inhibited HSP90, with IC<sub>50</sub> values of 0.78, 3.58, and 2.70 μM, respectively. The cell viability assay indicated that 4 inhibited all of the tested cancer cells proliferation, with a GC<sub>50</sub> value of 2.3 μM against NCIH1975 cell, but has little effect on human normal lung cell BEAS-2B cell. In the NCIH1975 xenograft models, 4 displayed strong antitumor activities at a dose of 10 and 20 mg/kg, but with no negative effect on the mice weight. In addition, 4 decreased the ECAR and lactate formation, increased OCR and ROS level in NCIH1975 cancer cell, which could be used as a promising modulator to reprogram the glucose metabolic pathways in NCIH1975 cancer cells.