Broccoli (Brassica oleracea var. italica) heads are commonly consumed in the Western diet and frequent consumption is thought to help protect against certain cancers and cardiovascular disease. Broccoli heads contain relatively high levels of glucosinolates and flavonols, thought to be the key phytochemicals that contribute to the health benefits gained upon consumption. In this study, we investigated the effect of applied nitrogen (N) at either 0, 15, 30 or 60 kg/ha, or 30, 60, 90 or 150 kg/ha with applied sulfur (S) at 50 or 100 kg/ha on the glucosinolates glucoraphanin, glucobrassicin and progoitrin, and the flavonols quercetin and kaempferol in broccoli cv. Marathon florets. Trials were conducted in two sites in either heavy clay or sandy loam to also assess the effect of soil type on phytochemical content. Application rates were based around recommended N and S applications for this crop in south-east Australia. N applications over 30 kg/ha caused a decrease in the content of glucoraphanin (18–34%) and both flavonols (20–38%). Progoitrin content was not affected while glucobrassicin increased by up to 44% with N applications >30 kg/ha. S applications of 50 or 100 kg/ha had no significant effect on either glucosinolates or flavonols. Crop yield (fresh weight), however, was significantly depressed (up to 40%) by N applications below 60 kg/ha. Fresh weight was also significantly depressed in plants grown in heavy clay compared with plants grown in a sandy loam, and phytochemical content increased, possibly due to a concentration effect. Therefore, low N applications to optimise phytochemicals may be only commercially useful if growers are producing mini-broccoli heads, as levels required to optimise phytochemical content (<30 kg/ha) also caused a significant decline in yield.