Microbial populations have evolved intricate networks of negotiation and communication through which they can coexist in natural and host ecosystems. The nature of these systems can be complex and they are, for the most part, poorly understood at the polymicrobial level. The Pseudomonas Quinolone Signal (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ) are signal molecules produced by the important nosocomial pathogenPseudomonas aeruginosa. They are known to modulate the behaviour of co-colonizing bacterial and fungal pathogens such asBacillus atropheaus,Candida albicansandAspergillus fumigatus. While the structural basis for alkyl-quinolone signalling withinP. aeruginosahas been studied extensively, less is known about how structural derivatives of these molecules can influence multicellular behaviour and population-level decision-making in other co-colonizing organisms. In this study, we investigated a suite of small molecules derived initially from the HHQ framework, for anti-virulence activity against ESKAPE pathogens, at the species and strain levels. Somewhat surprisingly, with appropriate substitution, loss of the alkyl chain (present in HHQ and PQS) did not result in a loss of activity, presenting a more easily accessible synthetic framework for investigation. Virulence profiling uncovered significant levels of inter-strain variation among the responses of clinical and environmental isolates to small-molecule challenge. While several lead compounds were identified in this study, further work is needed to appreciate the extent of strain-level tolerance to small-molecule anti-infectives among pathogenic organisms.