Nanoparticles, due to their extensive production and application, can have significant consequences for the environment, including soil and plant pollution. Therefore, it is very important to assess how nanoparticles will affect plants depending on the exposure pathways. The effect of gold nanoparticles in a concentration range of 1–100 mg/L on Mentha spicata L. during a 28-day experiment was investigated. Two routes of nanoparticles exposure were applied: root and foliar. Transmission electron microscopy was used to characterize nanoparticles and their effect on plant leaves’ ultrastructure. Gold content in soil and plant segments was determined using k0-neutron activation analysis. For root exposure, gold was mainly accumulated in soil (15.2–1769 mg/kg) followed by root systems (2.99–454 mg/kg). The maximum accumulation of gold in leaves (5.49 mg/kg) was attained at a nanoparticle concentration of 100 mg/L. Foliar exposure resulted in the maximum uptake of gold in leaves (552 mg/kg) and stems (18.4 mg/kg) at the highest applied nanoparticle concentration. The effect of nanoparticles on the Mentha spicata L. leaves’ biochemical composition was assessed. Nanoparticles affected the content of chlorophyll and carotenoids and led to an increase in antioxidant activity. Root exposure to gold nanoparticles resulted in an increase in the number of starch grains in chloroplasts and also suppressed the activity of the soil microbiota. Gold extraction from mint leaves into herbal infusion varied from 2 to 90% depending on the concentration of nanoparticles in the solution and the exposure route. The health risk as a result of gold exposure via herbal tea intake was assessed through estimated daily intake. The hazard quotient values were found to be less than the cutoff, indicating that a cup of tea infusion should not cause a serious impact to human health.