This study evaluated the efficacy of the yeast Pichia membranaefaciens alone or in combination with chitosan in controlling anthracnose in citrus fruits. The possible mechanisms were also analyzed. Results showed that the combination of P. membranaefaciens (1 × 108 cells/mL) and chitosan (0.01%, w/v) was better than the yeast alone in inhibiting Colletotrichum gloeosporioides infections. P. membranaefaciens which exhibited an antagonistic activity, attached to the hyphae of C. gloeosporioides and competed for space and nutrients. P. membranaefaciens secreted hydrolytic enzymes, such as chitinase and β-1,3-glucanase in Lilly–Barnett minimal salt medium supplied with cell wall preparation of C. gloeosporioides as the sole carbon source. Moreover, the yeast could survive with chitosan at a relative low concentration in vivo and in vitro. Chitosan inhibited spore germination and mycelial growth of C. gloeosporioides, and it induced marked morphological and structural changes of the pathogen. The beneficial effect of chitosan on enhancing the biocontrol activity of P. membranefaciens in inhibiting the anthracnose of citrus fruits is a result of the interactions among chitosan, P. membranaefaciens, C. gloeosporioides, and the citrus fruits