Significance Bacterial drug resistance is a crucial threat to global health, and antimicrobials with novel mechanisms of action are urgently needed. Antimicrobial peptides are natural molecules that kill bacteria mostly by perturbing their membranes, and they constitute promising compounds for fighting resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here, we show that the minimum peptide concentration needed for bacterial growth inhibition can vary by more than 100-fold with an increase in the density of cells in the initial inoculum of the assay (a phenomenon termed the “inoculum effect”). These findings question the utility of the currently used activity screening assays.