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In our continuous search for antibacterial agents against <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> (Psa) from kiwi-associated fungi, two pairs of epimeric cytochalasins, zopfiellasins A–D (<b>1</b>–<b>4</b>), were characterized from the fungus <i>Zopfiella</i> sp. The structures were established on the basis of spectroscopic data analysis, while the absolute configurations were determined by single-crystal X-ray diffraction. Compounds <b>1</b> and <b>3</b> exhibited antibacterial activity against Psa with MIC values of 25 and 50 μg/mL, respectively. This is the first report of anti-Psa activity of cytochalasin derivatives.