Historical processes in community assembly, such as the arrival order of species, strongly influence their interactions causing priority effects. Candida albicans and Pseudomonas aeruginosa are frequently co-isolated from biofilm-based infections of the skin, lungs, and medical devices. Their relationship, predominantly antagonistic, involves physical and chemical interactions. However, the presence and implications of priority effects among these microorganisms remain largely unexplored. Here, we developed dual biofilms with differing inoculation times for each species and assessed the resulting microbial communities' in vitro virulence and susceptibility. Our findings showed that the inoculation order impacts biofilm composition, structure, virulence, and antimicrobial susceptibility. The first colonizer had an advantage for surface colonization. Consecutive colonization increased biofilm virulence and negated C. albicans’ protective effect on P. aeruginosa PAET1 against meropenem treatment. Finally, we propose N-acetylcysteine as an adjuvant for treating C. albicans and P. aeruginosa interkingdom infections, working independently of priority effects.