Susceptibility testing was used to evaluate potential spectrum of action for piperazinyl–cross-linked fluoroquinolone dimers against test strains of Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and vancomycin-resistant Enterococcus faecium (VRE) and to evaluate dimers against fluoroquinolone-resistant and fluoroquinolone-susceptible strains of streptococci. Individual dimers displayed equivalent or lowered MIC values compared with parent fluoroquinolone monomers against test strains of S. pneumoniae, S. pyogenes, E. coli, and VRE. Raised MIC values were observed for all dimers in comparison to monomers against test strains of P. aeruginosa and E. coli. In comparison to parent fluoroquinolones, all dimers displayed decreased percent inhibition of growth against M. tuberculosis. Structural requirements for activity of dimers and partial dimers against all organisms, including lower MICs against certain fluoroquinolone-resistant and fluoroquinolone-susceptible strains of streptococci, were consistent with requirements previously observed for dimers against fluoroquinolone-susceptible and fluoroquinolone-resistant strains of S. aureus. In contrast, the 10- to 100-fold lowering of MICs against wild-type and fluoroquinolone-resistant strains of S. aureus previously observed for individual cross-linked dimers was not observed with test strains of the various organisms used here.