Violacein is a water-insoluble purple pigment produced by several gram-negative bacteria, including Janthinobacterium lividum. This pigment exhibits a wide range of biological activities, including anticancer, bacteriostatic, antibiotic, antifungal, antiparasitic, and antiviral properties. However, due to its low production yield, industrial-scale production has not yet been achieved. The main aim of the present study was to apply the Adaptive Laboratory Evolution (ALE) approach based on the basic principles of Darwinian evolution to enhance violacein production in J. lividum. Initially, the effects of various concentrations of ampicillin on bacterial growth and violacein production were evaluated. Subsequently, ALE was performed through sequential transfer in Erlenmeyer flasks containing different concentrations of ampicillin (0.5, 0.6, 0.7, 0.8, 0.9, 1 mg/mL). Twenty passages were conducted at each concentration, and the adapted strains were preserved in glycerol. After the final passage, cultures were transferred to agar plates containing ampicillin, and antibiotic-resistant colonies were selected for the next round. To investigate the effect of ampicillin on violacein and its precursor, the amino acid L-tryptophan (L-Trp), High-Performance Liquid Chromatography (HPLC) was employed. The results showed that violacein production increased significantly under sub-lethal concentrations of ampicillin; in the presence of 0.4 mg/mL of this antibiotic, violacein yield rose from 56 mg/L to approximately 132 mg/L. Through ALE, an evolved strain resistant to 1 mg/mL ampicillin was obtained, capable of producing 420 mg/L of violacein. Moreover, L-tryptophan production increased significantly compared to the control condition (from 0.62 μg/mL to 0.95 μg/mL). Overall, the results suggest that ampicillin may enhance violacein biosynthesis by stimulating the production of its precursor, L-tryptophan.