Pseudomonas aeruginosa is known to coexist and interact with Candida albicans in the polyspecies biofilms. In our previous work, treatment with the combined antimicrobials has been shown to significantly inhibit this polyspecies biofilm; however, the proteomic changes associated with this biofilm inhibition remain not well understood. Thus, the present work was performed to determine the proteome profile of P. aeruginosa-C. albicans polyspecies biofilms following treatment with the combined antimicrobials. The P. aeruginosa-C. albicans polyspecies biofilms were developed in 6-well microplates at 37 °C for 24 h under aerobic and anaerobic conditions. Following the treatment with 40 mg/ml combined antimicrobials (20 mg/ml erythromycin:20 mg/ml nystatin), whole-cell proteomes from the polyspecies biofilms were analyzed by a combination of two-dimensional polyacrylamide gel electrophoresis (2D SDS-PAGE) and mass spectrometry. On the other hand, the differentially expressed proteins were analyzed using STRING database. Results demonstrated that 3 and 9 P. aeruginosa proteins were differentially expressed in treated polyspecies biofilms under aerobic and anaerobic conditions, respectively. Based on the protein interaction network, several biological pathways such as carbohydrate metabolism, protein metabolism, and secondary metabolite metabolism in P. aeruginosa might be affected by the treatment with combined antimicrobials. There were no differentially expressed C. albicans proteins identified herein. It is possible that the inhibition of the polyspecies biofilms by the combined antimicrobial is associated with multiple biological pathways. The combined antimicrobials used in the present study may be useful to manage the diseases caused by P. aeruginosa-C. albicans polyspecies biofilms.