This study investigated the potential inhibitory effects of nine novel synthesized urea-substituted 1,4- dihydropyridine derivatives (DT-DEN-1-9) on polyphenol oxidase (PPO) activity. The compounds were synthesized via the Hantzsch reaction, providing a series of structurally diverse urea and thiourea-modified 1,4-dihydropyridines. Polyphenol oxidase enzyme was extracted from banana (Musa cavendishii) and purified using affinity chromatography with a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity gel. The purified enzyme's activity was measured spectrophotometrically using catechol as the substrate, monitoring the increase in absorbance at 420 nm. The inhibitory effects of the synthesized compounds on PPO activity were evaluated through in vitro assays. Various concentrations of each compound were incorporated into the enzyme reaction mixture, and the residual PPO activity was determined. The percentage of PPO activity was calculated relative to a control reaction without inhibitors. IC50 values, representing the concentration of inhibitor required to reduce enzyme activity by 50%, were determined using Lineweaver-Burk plots. Among the tested compounds, DT-DEN- 6, featuring a phenyl thiourea substituent, exhibited the most potent inhibition with an IC50 value of 100.14 μM. DT-DEN-8, containing a 2,5-dichlorophenyl thiourea moiety, also showed strong inhibitory activity with an IC50 below 150 μM. Structure- activity relationships were observed, with electron-withdrawing substituents generally enhancing inhibitory potency. Conversely, DT-DEN-5, bearing a 4-(trifluoromethyl)phenyl thiourea substituent, exhibited the weakest inhibition profile (IC50: 233.33 μM). Our findings provide valuable insights for the design of next-generation PPO inhibitors, potentially leading to the development of novel anti-browning agents for applications in food preservation and other industries where control of enzymatic browning is crucial.