This study was designed to analyze the effect of the mitochondrial respiratory pathways of Candida albicans (C. albicans) on the biofilm formation. The 2, 3-bis (2-methoxy- 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay was used to measure the metabolic activities of biofilms formed by the C. albicans which were cultured in the presence of respiratory pathways inhibitors. The biofilms formed by the wide type (WT), GOA7-deleted (GOA31), GOAV-reconstituted (GOA32), AOXla-deleted (AOX1) and AOXlb-deleted (AOX2) C. albicans strains were examined by the XTT reduction assay and fluorescence microscopy. The expression of adhesion-related genes BCR1, ALS1, ALS3, ECE1 and HWP1 in the biofilms formed by the above five C. albicans strains was detected by real time polymerase chain reaction. It was found that the metabolic activity of biofilms formed by C. albicans was decreased in the presence of alternative oxidase inhibitor whereas it was increased in the presence of classical mitochondrial respiratory pathway complex HI or complex IV inhibitor. AOX1 strain produced scarce biofilms interspersed with few hyphal filaments. Moreover, no significant changes in the expression of BCR1 and ALS3 were observed in the AOX1 strain, but the expression of ALSI and ECE1 was down-regulated, and that of HWP1 was up-regulated. These results indicate that both AOX1 and AOX2 can promote the biofilm formation. However, AOXla primarily plays a regulatory role in biofilm formation in the absence of inducers where the promoting effect is mainly achieved by promoting mycelial formation.