Exposure of vegetable seedlings to lowered temperature affects their growth and the content of certain chemical constituents. Chilling activates defensive mechanisms against stress and leads to acclimatization which increases plant ability to withstand harsh field conditions. Thus, seedlings subjected to low-temperature treatment had altered metabolic pathways, and these changes can persist until harvest. We therefore assessed: (i) the direct response of broccoli seedlings to 1 week or 2 weeks at lowered temperatures (6 °C, 10 °C, 14 °C, and 18 °C—control); and (ii) the long-term effects of the latter treatments on phytochemical components level in mature heads of broccoli cultivated in the field. Chilling stress decreased seedling shoot and root fresh and dry weights, plant height, number of leaves, leaf area, leaf perimeter, and leaf width. The most spectacular reductions in these parameters were observed at 6 °C and 10 °C. Longer exposure to lowered temperature resulted in greater reduction in the values of morphological traits. Chilling led to reduction in L-ascorbic acid content in broccoli seedlings, while a 6 °C temperature caused an increase in soluble sugars. The highest content of dry weight, soluble sugars, and L-ascorbic acid were observed in the heads of plants exposed to 14 °C; however, the content of dry weight (at 10 °C) and L-ascorbic acid (at 6 °C and 10 °C) also increased in broccoli heads in comparison with the control. Longer chilling (for 2 weeks) generally increased the content of these constituents in mature broccoli. Lower temperature (6–14 °C) applied at seedling stage increased P and Zn contents in broccoli heads in comparison to the control, whereas plants treated with 10 °C had more K, Fe, and, together with 6 °C treatment, Cu. The lowest temperature applied to the seedlings (6 °C) caused an increase in Mn content, while no effects of seedling chilling was noted for Ca levels. Significant linear correlations were noted and regression models were developed for the content of dry weight, soluble sugars, and L-ascorbic acid in the broccoli heads based on the chosen seedling parameters. The results show that the effect of lowered temperature to which the seedlings were subjected persists also in the further stages of plant ontogenesis, causing permanent changes in the chemical composition of mature broccoli heads.