Pseudomonas aeruginosa is a Gram-negative, multidrug-resistant opportunistic pathogen and a major cause of chronic lung infections in people with cystic fibrosis (CF). The CF lung environment contains thick, dehydrated mucus that limits oxygen penetration, and the formation of P. aeruginosa biofilms within hypoxic mucus plugs enhances this organism’s antimicrobial tolerance. Identifying pathways that regulate P. aeruginosa biofilm formation is therefore critical for developing new CF therapeutics. Biofilm formation in P. aeruginosa is strongly influenced by iron availability, as iron is essential for numerous cellular processes. The host innate immune system limits pathogen access to iron and other transition metal ions by secreting metal-sequestering proteins, a defense known as nutritional immunity. Most studies of nutritional immunity have been performed under aerobic conditions, where the ferric ion [Fe(III)] predominates, while few have explored nutritional immunity in the context of anoxic environments, where the ferrous ion [Fe(II)] is dominant. Calprotectin (CP) is the only known host protein capable of sequestering Fe(II) and also exhibits functional diversity via its ability to bind iron, zinc, manganese, and nickel. CP is abundant in the lungs of individuals with CF and has been termed the CF antigen. Given the high abundance of CP, low oxygen levels, and increased Fe(II) in CF lungs, understanding how CP affects P. aeruginosa under anaerobic and biofilm conditions is essential for elucidating CF disease progression. This work investigates the antimicrobial activity, iron starvation responses, and phenotypic changes of P. aeruginosa in response to CP under anaerobic conditions and within flow cell biofilms. Whole transcriptome analysis combined with biochemical and phenotypic assays revealed that CP induces an iron starvation response in anaerobic conditions that shares similarities with, but remains distinct from, the response observed in aerobic environments. Furthermore, the use of an optimized flow cell biofilm system and fluorescent genetic reporter strains, demonstrated that CP exhibits antimicrobial activity against P. aeruginosa biofilms through both metal-dependent and metal-independent mechanisms. Overall, this work expands our understanding of how host nutritional immunity influences iron homeostasis and biofilm physiology in P. aeruginosa, providing new insights into the complex host–pathogen interactions that drive chronic CF lung infections.