Low temperatures are a substantial limitation in the geographic distribution of warm-season crops such as cucumber (Cucumis sativus L.). Tolerance to low temperatures varies among different plant species and genotypes when changes in environmental cues occur. Therefore, biochemical and biophysical events should be coordinated to form a physiological response and cope with low temperatures. We examined how light intensity influences the effects of low temperature on photosynthesis and some biochemical traits. We used chlorophyll fluorescence imaging and polyphasic fluorescence transient to analyze cold stress damage by 4 °C. Photosynthetic Photon Flux Densities (PPFDs) of 0, 300, and 600 μmol m⁻² s⁻¹, in four accessions of cucumber, were investigated. The results show that the negative effects of cold stress are PPFD-dependent. The adverse effect of cold stress on the electron transport chain is more pronounced in plants exposed to 600 μmol m⁻² s⁻¹ than the control and dark-exposed plants, indicated by a disturbance in the electron transport chain and higher energy dissipation. Moreover, biochemical traits, including the H₂O₂ content, ascorbate peroxidase activity, electrolyte leakage, and water-soluble carbohydrate, increased under low temperature by increasing the PPFD. In contrast, chlorophyll and carotenoid contents decreased under low temperature through PPFD elevation. Low temperature induced a H₂O₂ accumulation via suppressing ascorbate peroxidase activity in a PPFD-dependent manner. In conclusion, high PPFDs exacerbate the adverse effects of low temperature on the cucumber seedlings.