Ribonuclease 7 (RNase 7) is a 14.5-kDa peptide that possesses potent antimicrobial properties against Gram-negative and Gram-positive bacteria and is expressed in a variety of epithelial tissues. Little is known about its mechanisms of action and the determinants of its antimicrobial properties. The objective of this study was to identify the intrinsic functional domains of RNase 7 that influence its activity against uropathogenic bacteria. A series of RNase 7 fragments were generated that contained different components of its secondary motifs starting from both the N terminus and the C terminus of RNase 7. We determined the antimicrobial properties of each fragment against both Gram-positive Staphylococcus saprophyticus and Gram-negative Escherichia coli and Proteus mirabilis. RNase 7 fragments displayed significant differences in their antimicrobial activity profiles. Compared to N-terminal fragments, C-terminal fragments showed uniformly decreased activity against Gram-negative E. coli and P. mirabilis and Gram-positive S. saprophyticus. Fragments that lack β-sheets 1, 3, and 4 also demonstrated significantly decreased activities. We have also identified one fragment with at least 4-fold increased potency against both E. coli and Staphylococcus compared to full-length peptide. We identified distinct regions of the peptide that are independently responsible for Gram-negative and Gram-positive activity. Our results suggest that distinct mechanisms are responsible for RNase 7's antimicrobial activity against various uropathogens.