Although Rhodiola crenulata (R. crenulata) exhibits anti-tumor effects, its mechanism of action has yet to be elucidated. Lactate dehydrogenase (LDH), a key glycolytic enzyme in tumor metabolism, has emerged as a promising target for anticancer drug development. To elucidate the anticancer mechanism of R. crenulata, this study systematically screened its bioactive components for inhibitory activity against LDHA and LDHB subunits. First, the main components of R. crenulata were identified using HPLC-QTOF MS. Then, the inhibitory potency of these identified components was assessed against recombinant LDHA and LDHB. Finally, isoenzyme inhibition of the bioactive components was elucidated through structure-based molecular docking and cell viability assays. The results showed that five R. crenulata compounds—quercetin, luteolin, kaempferol, epicatechin gallate, and ellagic acid—showed significant LDH inhibition, with stronger effects on LDHA than on LDHB. Against LDHA, the IC₅₀ values for quercetin, luteolin, kaempferol, and epicatechin gallate were 0.27 ± 0.02 µM, 1.19 ± 0.09 µM, 0.70 ± 0.13 µM, and 2.27 ± 0.23 µM, respectively. Against LDHB, the values for quercetin, luteolin, and kaempferol were 0.87 ± 0.07 µM, 2.71 ± 0.39 µM, and 8.69 ± 0.85 µM, respectively. Molecular docking simulations and cell viability assays of the five bioactive compounds revealed their interactions with LDH subunits and supported their inhibitory effects. This study provides the first comprehensive inhibition profile of R. crenulata targeting LDH isoenzymes. It underscores the potential of R. crenulata in LDH-targeted therapeutics and supports its further development for cancer treatment.