Bush tea is a popular South African herbal and medicinal tea with the potential for commercialization. The objective of the study was to investigate the effects of different rates of applied nitrogen (N) and timing (early and late) of N application on yield, chemical composition, pharmacologic activity, and cytotoxicity of bush tea. Factorial treatments consisted of timed N application rates (0, 75, 150, and 225 kg·ha–1) for both early and late N application. The treatments were arranged in a randomized complete block design with five replicates. The results show a significant positive response of bush tea fresh and dry leaf and twig weight, chlorophyll, leaf tissue N, total polyphenols, and total flavonoids in response to applied N rates, regardless of the timing of N application, reaching a maximum at 225 kg·ha–1 of N. Later, regardless of the timing of N application, total tannins and total antioxidant activity increased with increasing applied N from 0 to 225 kg·ha–1, reaching a maximum of 150 kg·ha–1 N. The results of this study suggest that, with the exception of antimicrobial activity, most of the parameters recorded increased with increasing rates of N applied. The results further denote that parameters recorded were consistently greater on early applied N compared with late applied N. However, there was no significant difference between the minimum inhibition concentration (MIC) and minimum microbicide concentration (MMC) of early and late applied N. Bush tea applied with 75 N and 150 N had a significant MIC value (3.1 mg·mL–1) for Escherichia coli and Klebsiella pneumonia compared with 6.3 mg·mL–1 reached at 225 kg·ha–1 N. Among N rates applied, the MIC for Klebsiella oxytoca, Proteus vulgaris, Salmonella typhi, Serratia marcescence, and Staphylococcus aureus were not significantly different. Hence, N rates applied did not have a significant effect on bush tea MMC values of all microbial species tested. The cytotoxicity of bush tea leaf and twigs harvested from early and late N application were significantly reduced with increasing nitrogen levels reaching a maximum at 225 kg·ha─1. There was a wide variation of compounds despite rates of N applied as well as timing of application, with most compounds such as norfenfluramine, phytol, caryophyllene, propylene glycol, α-copaene, and squalene detected in greater quantities.