Broccoli is a source of bioactive compounds that provide an important nutritional value. The content of these compounds can vary depending on agronomic and environmental conditions, as well as on elicitation. In this study, three crop trials were carried out to evaluate the effects of the cultivation season, the application of different dosages of methyl-jasmonate (MeJA) on the overall quality and on the total content of bioactive compounds of ‘Parthenon’ broccoli cultivated under the field conditions of southeastern Spain. Color parameters, chlorophyll content, total phenolic compounds, total flavonoids and antioxidant activity were measured to evaluate the overall quality. Moreover, individual carotenoids, phenolic compounds and glucosinolates were evaluated by high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography equipped with diode array detector coupled to mass spectrometer using electro spray ionization (HPLC-DAD-ESI/MSn). The content of total carotenoids, phenolic compounds and glucosinolates were higher in autumn compared with spring, showing increases of 2.8-fold, 2-fold and 1.2-fold, respectively. Moreover, a double application of MeJA increased the contents of total carotenoids, phenolic compounds and glucosinolates by 22%, 32% and 39%, respectively, relative to the untreated samples. Considering our results, the controlled and timely application of 250 µM MeJA to the aerial parts of the plants four days before harvest, on two consecutive days, seems to be a valid agronomic strategy to improve the health-promoting capacity of Parthenon broccoli, without compromising its overall quality.