Amphotericin B (AMB, <b>1</b>) is the most powerful antibiotic in treating potentially life-threatening invasive fungal infections (IFIs), though severe toxicity derived from self-aggregation greatly limits its clinical application. Herein, we applied a bisamidation strategy at the C16-COOH and C3'-NH<sub>2</sub> to improve the therapeutic properties by suppressing self-aggregation. It was found that basic amino groups at the residue of C16 amide were beneficial to activity, while lipophilic fragments contributed to toxicity reduction. Additionally, <i>N</i>-methyl-amino acetyl and amino acetyl moieties at C3' amide could help keep the fungistatic effectiveness. The modification work culminated in the discovery of <b>36</b> (ED<sub>50</sub> = 0.21 mg/kg), which exerted a 1.5-fold stronger antifungal efficacy than amphamide, the optimal derivative theretofore, in mice, low self-aggregation propensity, and thus low acute toxicity. With the improvement in therapeutic index and good PK profile, <b>36</b> is promising for further development as a second-generation polyene antifungal agent.