Baby spinach is becoming increasingly popular as a salad ingredient and needs high fertiliser rates to grow well and attain higher-quality leaves (dark green leaves). Chemical fertilisers, especially nitrogen (N), boost yields. There are many risks associated with nitrogen fertilisation. Additionally, spinach contains phenolic compounds and carotenoids. Nitrogen fertilisation affects growth, development, yield and metabolites. This study examined the impact of lower concentrations of N (0, 30, 60, 90, 120, 150 mg/L) on yield and colour properties [light intensity (L*) colour coordinates, unique for green colour (a*) and yellow colour (b*)], as well as the impact of varying N concentrations on the total phenolic content and p-coumaric acid, quercetin, ferulic acid, kaempferol, lutein, zeaxanthin, β-carotene and antioxidant activities in the baby spinach varieties 'Acadia', 'Crosstrek' and 'Traverse', and it was established that N fertilisation improves phytochemical bioaccessibility and antioxidant activity. In a split strip plot design, three baby spinach varieties were treated with different N concentrations, including 0, 30, 60, 90, 120 and 150 mg/L. For 40 days, three baby spinach varieties were grown on soilless Mikskaar Professional substrate 300. During both seasons, 'Crosstrek' had the highest fresh mass (921.4 g/m2, 856.3 g/m2) at 120 mg/L N, while 'Traverse' had the highest fresh mass at 554.8 g/m2 and at 564.3 g/m2 at 90 mg/L N and did not differ significantly from 90 to 150 mg/L N during either season. During both seasons, 'Acadia' at 90 mg/L N increased fresh mass to 599 g/m2 and 557.9 g/m2. The variety × N supply interaction significantly affected the leaf colour; chlorophyll content across seasons; the levels of bioactive compounds, p-coumaric acid, quercetin, ferulic acid, kaempferol, lutein, zeaxanthin and β-carotene in spinach varieties; the in vitro bioaccessibility; and the antioxidant activity. Varietal differences influenced the bioaccessibility of phenolic compounds and carotenoid components. The appropriate N levels can be used during plant cultivation to optimise the bioaccessibility of this spinach variety. Thus, fertilising 'Traverse' with 90 mg/N mL increased the in vitro bioaccessibility of β-carotene (35.2%), p-coumaric acid (7.13%), quercetin (8.29%) and ferulic acid (1.92%) without compromising the yield.