L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is a non-standard amino acid, and the gold standard drug for the treatment for Parkinson’s Disease (PD). Recently, a gene encoding the enzyme that is responsible for its synthesis, as a precursor of the coloured pigment group betalains, was identified in beetroot, BvCYP76AD6. We have engineered tomato fruit enriched in L-DOPA through overexpression of BvCYP76AD6 in a fruit specific manner. Analysis of the transgenic fruit revealed the feasibility of accumulating L-DOPA in a non-naturally betalain-producing plant. Fruit accumulating L-DOPA also showed major effects on the fruit metabolome. Some of these changes included elevation of amino acids levels, changes in the levels of intermediates of the TCA and glycolysis pathways and reductions in the levels of phenolic compounds and nitrogen-containing specialised metabolites. Furthermore, we were able to increase the L-DOPA levels further by elevating the expression of the metabolic master regulator, MYB12, specifically in tomato fruit, together with BvCYP76AD6. Our study elucidated new roles for L-DOPA in plants, because it impacted fruit quality parameters including antioxidant capacity and firmness. The L-DOPA levels achieved in tomato fruit were comparable to the levels in other non-seed organs of L-DOPA – accumulating plants, offering an opportunity to develop new biological sources of L-DOPA by widening the repertoire of L-DOPA-accumulating plants. These tomato fruit could be used as an alternative source of this important pharmaceutical.