ABSTRACTIn this study, we have focused on a multiparametric microbiological analysis of the antistaphylococcal action of the iodinated imine BH77, designed as an analogue of rafoxanide. Its antibacterial activity against five reference strains and eight clinical isolates of Gram-positive cocci of the generaStaphylococcusandEnterococcuswas evaluated. The most clinically significant multidrug-resistant strains, such as methicillin-resistantStaphylococcus aureus(MRSA), vancomycin-resistantS. aureus(VRSA), and vancomycin-resistantEnterococcus faecium, were also included. The bactericidal and bacteriostatic actions, the dynamics leading to a loss of bacterial viability, antibiofilm activity, BH77 activity in combination with selected conventional antibiotics, the mechanism of action,in vitrocytotoxicity, andin vivotoxicity in an alternative animal model,Galleria mellonella, were analyzed. The antistaphylococcal activity (MIC) ranged from 15.625 to 62.5 μM, and the antienterococcal activity ranged from 62.5 to 125 μM. Its bactericidal action; promising antibiofilm activity; interference with nucleic acid, protein, and peptidoglycan synthesis pathways; and nontoxicity/low toxicityin vitroandin vivoin theGalleria mellonellamodel were found to be activity attributes of this newly synthesized compound. In conclusion, BH77 could be rightfully minimally considered at least as the structural pattern for future adjuvants for selected antibiotic drugs.IMPORTANCEAntibiotic resistance is among the largest threats to global health, with a potentially serious socioeconomic impact. One of the strategies to deal with the predicted catastrophic future scenarios associated with the rapid emergence of resistant infectious agents lies in the discovery and research of new anti-infectives. In our study, we have introduced a rafoxanide analogue, a newly synthesized and described polyhalogenated 3,5-diiodosalicylaldehyde-based imine, that effectively acts against Gram-positive cocci of the generaStaphylococcusandEnterococcus. The inclusion of an extensive and comprehensive analysis for providing a detailed description of candidate compound-microbe interactions allows the valorization of the beneficial attributes linked to anti-infective action conclusively. In addition, this study can help with making rational decisions about the possible involvement of this molecule in advanced studies or may merit the support of studies focused on related or derived chemical structures to discover more effective new anti-infective drug candidates.