Many herbs and spices have been shown to contain high levels of polyphenolic compounds with potent antioxidant properties. In the present study, we explore how nutrient availability, specifically nitrogen fertilization, affects the production of polyphenolic compounds in three cultivars (Dark Opal, Genovese, and Sweet Thai) of one of the most common culinary herbs, basil (Ocimum basilicum L.). Nitrogen fertilization was found to have a significant effect on total phenolic levels in Dark Opal (p < 0.001) and Genovese (p < 0.001) basil with statistically higher phenolic contents observed when nutrient availability was limited at the lowest (0.1 mM) applied nitrogen treatment. Similarly, basil treated at the lowest nitrogen fertilization level generally contained significantly higher rosmarinic (p = 0.001) and caffeic (p = 0.001) acid concentrations than basil treated at other nitrogen levels. Nitrogen fertilization also affected antioxidant activity (p = 0.002) with basil treated at the highest applied nitrogen level, 5.0 mM, exhibiting lower antioxidant activity than all other nitrogen treatments. The anthocyanin content of Dark Opal basil was not affected by applied nitrogen level, but anthocyanin concentrations were significantly impacted by growing season (p = 0.001). Basil cultivar was also determined to have a statistically significant effect on total phenolic levels, rosmarinic and caffeic acid concentrations, and antioxidant activities.