A series of 7-(3'-substituted)pyrrolidino-8-methoxyisothiazoloquinolone (ITQ) analogues were prepared, and their antibacterial potency against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were compared. Many of these analogues had MIC ≤ 0.25 μg/mL against quinolone-resistant MRSA strains. The stereochemical preference was explored for a series of 1''-methyl-3'-aminomethylpyrrolidine analogues. Antibacterial activity was generally more favorable with 3'-R, 1''-S configuration. Substitution on the 3'-aminomethyl nitrogen tended to decrease activity, while potency was maintained with disubstitution or aryl substitution at the 1''-carbon. The 7-[(R)-3-((S)-1-aminoethyl)pyrrolidin-1-yl] analogue (6a(R,S)) and the (R)-7-[3-(2-aminopropan-2-yl)pyrrolidin-1-yl] analogue (7a(R)) were found to be the ITQs with the most promising antibacterial profiles. The MICs of these select ITQs versus a panel of clinical MRSA strains were determined, and the ITQs were found to have 8- to 16-fold greater potency than linezolid. These analogues were also evaluated for inhibition of the target enzymes, topoisomerase IV and DNA gyrase, from both wild-type and multidrug resistant strains. The ITQs were up to >30 times more inhibitory against these targets than the fluoroquinolone moxifloxacin.