Microbial biofilms are difficult to tackle in many infectious diseases. Candida albicans and Staphylococcus aureus are prevalent symbiotic strains in polymicrobial biofilms, which showed enhanced antimicrobial resistance and made identifying effective treatment techniques more difficult. The antibiofilm abilities of tachplesin I analogue peptide (TP11A) and tachplesin I were investigated quantitatively in this study. Both inhibited C. albicans monomicrobial, S. aureus monomicrobial, and C. albicans−S. aureus polymicrobial biofilms quite well. TP11A suppressed the biofilm-and virulence-related genes of C. albicans (hwp 1) and S. aureus (ica A, f nb B, agr A, hla, nor A, and sig B) in the mixed biofilm, according to quantitative reverse transcription polymerase chain reaction analysis. We created an injectable thermosensitive in situ PLEL@TP11A gel system by simply adding TP11A into poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) (PLEL). Using C. albicans−S. aureus mixed infected wound models of mice, the in vivo therapeutic effect of TP11A and PLEL@TP11A in polymicrobial infections was investigated. The findings revealed that TP11A and PLEL@TP11A could efficiently reduce bacterial and fungal burden in wound infections, as well as accelerated wound healing. Based on above findings, TP11A might be an effective antimicrobial against C. albicans−S. aureus poly-biofilm formation and mixed infection.