A library of monosubstituted chalcones (<b>1</b>-<b>17</b>) bearing electron-donating and electron-withdrawing groups on both aromatic rings were selected. The cell viability on human tumor cell lines was evaluated first. The compounds unable to induce detectable cytotoxicity (<b>1</b>, <b>13</b>, and <b>14</b>) were tested using the monoamine oxidase (MAO) activity assay. Interestingly, they inhibit MAO-B, acting as competitive inhibitors, with <b>13</b> and <b>14</b> showing the best profiles. In particular, <b>13</b> exhibited a potency higher than that of safinamide, taken as a reference. Docking studies and crystallographic analysis showed that in human MAO-B <b>13</b> binds with the halogen-substituted aromatic ring in the entrance cavity, similar to safinamide, whereas <b>14</b> is accommodated in the opposite way. The main conclusion of this cell biology, biochemistry, and structural study is to highlights <b>13</b> as a chalcone derivative that is worth consideration for the development of novel MAO-B-selective inhibitors for the treatment of neurodegenerative diseases.