Globally, antibiotic resistance is a growing problem, reducing successful treatment of bacterial infections and increasing mortality. Antibiotic resistance mutations are typically associated with fitness costs, reducing competitive ability in the absence of antibiotics. These fitness costs are thought to represent a key barrier to the spread of resistance, but are traditionally characterised in laboratory-based assays, which lack the presence of other microbes; a key deviation from the biological environment of infection and transmission. Here, we investigated whether microbial interactions influence the existence and magnitude of fitness costs associated with antibiotic resistance mutations in the opportunistic bacterial pathogen, Pseudomonas aeruginosa. A large-scale screen of over 400 assays was used to characterise microbially-driven changes to the fitness costs of spontaneous resistance mutations, using a library of 33 resistant strains and 13 human-associated bacterial and fungal species. Microbial interactions led to significant (p < 0.01) changes in the fitness costs of antibiotic resistance mutations in ~10% of assays. Interactions were found to most frequently increase the fitness costs of aztreonam resistance-associated mutations, and mutations in protein biosynthesis genes were most frequently associated with microbially-driven fitness costs. Preliminary results suggest that context-dependent fitness effects are predominantly linked to microbial metabolism. Further investigations of the underlying mechanisms will help us better understand the drivers of these fitness impacts. Understanding factors that influence fitness costs of resistance is crucial for predicting how resistance mutations spread and, ultimately, may lead to novel strategies for combating resistance by manipulating these costs.