Microbial resistance inhibition is increasingly focused on the use of plant extracts and their phytochemicals as candidates for targeting virulence factors. Here, we report on the chemical composition and virulence inhibition potential of both polar (PF) and non-polar fractions (NPF) of the underground parts of three common Iris species (I. confusa, I. pseudacorus and I. germanica). The anti-haemolytic and biofilm inhibition potentials of the aforementioned Iris species on methicillin resistant and sensitive (MRSA and MSSA) S. aureus bacterial strains were explored. I. pseudacorus PF exhibited the most potent effect against S. aureus haemolytic activity. Intriguingly, all the tested fractions from all species, except I. pseudacorus NPF, have no significant inhibition on the biofilm formation of MRSA and MSSA. Metabolite profiling of the investigated species revealed ninety and forty-five metabolites detected in the PFs and NPFs, respectively. Nigricin-type, tectorigenin-type isoflavonids and xanthones allowed the discrimonation of I. pseudacorus underground parts PF from the other species, highlighting the importance of those metabolites in exerting its promising activity. On the other hand, triterpene acids, iridals, triacylglycerols, ceramides, and acid represented the metabolites detected in highest abundance in I. pseudacorus NPF.